KR101603632B1 - Use of human cells of myeloid leukaemia origin for expression of antibodies - Google Patents

Abstract

(A) introducing one or more nucleic acids encoding at least a portion of a protein in a host cell that is immortalized human blood cells; (b) culturing the host cell under conditions that allow production of the protein composition; And (c) separating the protein composition. The present invention also relates to a method for producing a protein molecular composition having a defined glycosylation pattern.

Description

USE OF HUMAN CELLS OF MYELOTIC LEUKEMIA FOR ANTIBODY EXPRESSION BACKGROUND OF THE INVENTION < RTI ID = 0.0 > [0001] <

The present invention provides protein molecular compositions having enhanced activity and / or increased yield and / or improved homogeneity and human glycosylation, and in particular, biotechnologically advantageous methods for producing antibody molecule compositions. The present invention further provides novel host cells, nucleic acid, and protein molecule compositions.

Important features and challenges in the industry are the generation of recombinant proteins and the important issues that still require optimization of productivity, cost, homogeneity and protein activity. Glycosylation is also an important problem in yielding high yields of these which raise a series of important problems for the production of homogeneous recombinant glycoproteins. Current producing cell lines provide a series of different challenges and problems due largely to the complexity of glycosylation and species, tissue and site specificity. Thus, optimization of the production system associated with glycosylation remains one of the important aspects for optimization. This has a significant effect on the activity, immunogenicity, bioavailability and half-life of the protein molecule, in particular as a difference in the glycosylation pattern. A detailed overview of the glycosylation properties of different cell lines and of different cell lines derived from non-mammalian production systems is provided in Jenkins et al (Getting the glycosylation right: implications for the biotechnology industry, Nat Biotechnology, 1996, 14: 975-981) .

Antibodies are a major tool for diagnosis and research and are likely to be the largest class of therapeutic agents. Therapeutics are on sale for 10 recombinant antibodies and hundreds of them are under clinical development. Important features and challenges in the industry are the generation of recombinant antibodies ("rMAb") and productivity, cost, homogeneity and antibody activity are still important issues to be optimized. Nearly all commercial therapeutic rMAbs were produced in rodent cell lines from hamsters (CHO) and mice (NS0 or Sp2 / 0). Most of the rMAbs developed are by far in rodent cells and others are under development, but none were sufficient to optimize productivity, cost, homogeneity and antibody activity.

Glycosylation is also an important problem in yielding a high yield of homogeneous and potentially rMAb which poses a series of important problems for the production of rMAb. Current producing cell lines provide a series of different challenges and problems due to the complexity of glycosylation and species, tissue and site specificity [Review: Royston Jefferis, CCE IX: Glycosylation of Recombinant Antibody Therapeutics; Biotechnol. Prog. 2005, 21, 11-16].

Thus, optimization of proteins associated with glycosylation and, in particular, antibody production systems remains one of the important aspects to be optimized.

The present invention provides a novel expression system based on immortalized human blood cells and, in particular, cells of myeloid leukemia origin. Such cells surprisingly improve the production of glycosylated proteins and especially antibodies in terms of activity, yield and homogeneity.

Proteins are a diverse group of functions and occurrences that vary greatly from one another. Of the proteins that have therapeutic potential, most proteins are glycosylated and are associated with a number of hormones such as growth hormone, glicagonone, FSH and LH, growth factors such as GM-CSF, G-CSF, VEGF, (E. G., Leuprorelin, decirudin), blood coagulation factors (e. G., Factor VII < / RTI > , VIII and IX), vaccines (e.g., hepatitis B antigens) and antibodies. Established cell generation systems are unable to produce native human glycosylated proteins. Prokaryotic (e. G., Bacterial) and most eukaryotic systems (e. G., Yeast, insect and plant cells) have glycans that synthesize proteins lacking glycosylation or are significantly different from human carbohydrate chains. Chinese hamster ovary (CHO) cells are a commonly used production system capable of glycosylating proteins in a manner similar to human cells. However, important problems remain in various aspects of, for example, galactosylation, fucosylation, especially glycosylation with N-acetylglucosamine, and in particular in various aspects of sialylation. These differences affect activity, bioavailability, immunogenicity and half-life.

At the time this generation system was established, it was sufficient to produce a therapeutically active protein that was at least somewhat active. However, much effort has been devoted to improving the activity of therapeutic proteins today to (i) reduce the frequency and concentration of applied doses of therapeutic proteins, (ii) reduce treatment costs, and (iii) reduce side effects .

A major strategy for improving the bioactivity of proteins is to lengthen their bioavailability by prolonging their serum half-life. This can be done, for example, by a process called pegylation, wherein certain forms of polyethylene glycol are chemically added / bound to the resulting protein. PEG increases molecular weight and therefore increases serum half-life. However, this process involves many problems. For example, in almost all cases, pegylation reduces the activity of a protein by its cell effector function and / or repeated administration in humans often leads to an adverse immune response that neutralizes the antibody and / Requiring additional chemical modifications, resulting in a multi-step process with additional cost, consumption and time. Similar carrier systems (e.g., HESylation or attachment of albumin) have comparable disadvantages.

Also the modification of the carbohydrate chain and thus the glycosylation of the protein is focused on improving the serum half-life of the protein expressed by recombination. This technique focuses on maximizing the degree of sialylation of the recombinant glycoprotein. Sialic acid is the most abundant terminal monosaccharide on the surface of eukaryotic cells, and it is generally believed that as more glycoproteins are sialylated their serum half-life is longer during circulation. This is based on the presence of a specific receptor as an asialo protein-receptor in the liver that binds to circulating sialylated proteins and leads them to degradative cells.

Various classes of antibodies are present in humans and most mammals, namely IgG, IgM, IgE, IgA, IgD. Although molecules of all antibody classes are used as diagnostic or research tools, most antibody-based therapeutics sold and under development are IgG and to a lesser extent, IgM. The human IgG class is further classified into four subtypes IgG1, IgG2, IgG3 and IgG4. The basic structure of an IgG molecule consists of two light and two heavy chains, each containing one variable domain comprising the antigen-binding site and two Fab domains with one constant domain, a further constant domain and a ligand One Fc region comprising the acting site, and a flexible hinge region connecting the Fab and Fc regions. The antibody may be present as a whole molecule or as an antibody fragment, such as a Fab fragment or a short chain Fv comprising two variable regions of heavy and light chains linked through a linker. Figure 18 shows IgG antibodies.

Therapeutic recombinant antibodies are generally chimeric, humanized or so-called complete human protein sequences to reduce their immunogenicity. However, virtually complete human molecules could not be readily obtained because the post-translational modification, particularly glycosylation, is not a human due to the production in rodents or CHO cells, and therefore such modifications as found in human antibodies to human serum It can be different. Differences in the modification, particularly the glycosylation pattern, can have a significant impact on the activity and immunogenicity of the individually generated antibodies.

The activity of the antibody may be affected or influenced by the combined performance. On the other hand, the antibody has a certain specificity mediated by the variable region ("V region") of the antibody located in the Fab region, the specific sequence of the V region, and the CDR region. They play an important role in determining the specificity and affinity of the antibody. Thus, the V region is critical for epitope binding properties and modifies antibodies and antibodies. The affinity of the antibody indicates the binding strength and kinetics of the single binding region of the antibody to the epitope. Since the various antibody classes and subgroups have two to ten identical variable domains in an antibody molecule, the binding strength and kinetics of the antibody are dependent on the number of binding sites available for binding to the epitope on the target antigen or on the cell, It is influenced by accessibility and is indicated by its antigen binding ability .

Another important factor for the quality of the antibodies used for diagnosis and in particular treatment is the number of binding sites of the antibody on the target structure or cell as well as its internalization rate. This quality in particular affects the effectiveness and suitability of the antibodies used in therapy.

On the other hand, the effector mechanism associated with the therapeutic use of the antibody is several fold, whereby some of the antibodies act through a single mechanism and others act by a combination of various effector mechanisms. One strategy is to couple an antibody or antibody fragment to an effector molecule that mediates a therapeutic effect, for example, by coupling an immune effector molecule, such as interleukin 2, for anti-tumor therapy, to mobilize the immune system, Target cells are killed by coupling isotopes. Radiolabeled antibodies or antibody fragments are also valuable for in vivo diagnosis.

Other strategies include non-labeled, so-called " naked "antibodies, which can have significant advantages such as the use of low toxicity, less complex logistic, natural immune effector arms or therapeutic effects that do not require additional effector molecules . A number of studies have been conducted to identify the mechanism and mode of action of antibodies and to develop antibodies using these activities. The most important activities and effects are the antibody-dependent cell-mediated cytotoxic activity (referred to herein as " ADCC activity "), complement-dependent cytotoxicity (Referred to herein as " CDC activity "), predation mediated cytotoxic activity (referred to herein as" predominant activity "), and receptor mediated activity (referred to herein as" receptor mediated activity ").

Receptor mediated activity is based primarily on binding of an antibody to a molecule or receptor on the target cell or inhibits binding of a particular molecule to a target cell to induce a specific effect such as antagonistic or potentiation activity on that cell or receptor blockade of the antibody Or suppressing the secretion or secretion of a particular molecule in a target cell or by inducing or inhibiting the apoptosis or proliferation of a target cell or by inhibiting the secretion or secretion of a specific molecule in a target cell or by administering certain other receptor mediators such as intercellular or intercellular interactions Based on blocking or initiating an incident. ADCC activity, CDC activity, and predatory activity are cytotoxic activities mediated by the Fc region of the antibody (referred to herein as " Fc partially mediated activity "), receptor mediated activity, affinity, The specificity is mediated primarily through the binding region of the antibody contained in the Fab region.

Fc partially mediated activity is coupled to effector ligands such as killer cells, natural killer cells and immunologic effector cells such as activated macrophages, or complementary sequences of Fc-gamma RI, Fc-gamma RII, Fc-gamma RIII, (FcRn), and the like. The human IgG1 subtype is reported to have the highest ADCC and CDC activity among the human IgG class molecules. For IgM, CDC activity is the predominant effector mechanism. ADCC activity and CDC activity include binding of the Fc region, which is a constant region of the antibody to an Fc-receptor such as Fc-gamma RI, Fc-gamma RII, Fc-gamma RIII or complementary sequence components such as C1q of effector cells. Important amino acid residues are in the hinge region and particularly in the second domain of the constant region ("C gamma 2 domain"). The carbon chain linked to the C gamma 2 domain is important for activity. The carbohydrate chain is bound to the amino acid asparagine 297 (Asn-297) of C-Gamma 2 (N-glycoside-linked carbohydrate chain). The carbohydrate chain end attached to asparagine is called the reducing end and the opposite end is called the non-reducing end. The Fc region of an IgG antibody has two carbohydrate binding sites. Figure 18 shows the structure of the IgG molecule and typically indicates the position at which a carbohydrate structure can be found. In addition, the chemical structure and composition of such carbohydrate structures are described.

From Fc partial mediated activity, two are expected to be affected by the glycosylation of the antibody: Elimination of the sugar chains in the Fc portion of the antibody results in the loss of CDC and ADCC activity, and in this N-glycan, galactose Reduction of CDC activity results in a decrease in CDC activity [Boyd et al., Molecular Immunol., 32, 1311, (1995); US6946292. In addition, the expression of the antibody in rat cells increases the ADCC activity of certain antibodies expressed therein compared to the same antibody expressed in hamster and rat cells [Lifely et al., Glycobiology, 1995 Dec; 5 (8): 813-22; WO00 / 61739]. Both reports assume that changes in glycosylation result in this difference in antibody ADCC activity, but this is not clear. Lifely et al. 1995] assumed that the bisecting N-acetylglucosamine ("bisecting GlcNAc") is responsible for the increased ADCC activity of the antibody, whereas WO00 / 61739 suggests that FCO binding to N-acetylglucosamine at the reducing end of N-glycans It is hypothesized that the dramatic decrease in OSa alpha 1-6 ("core-fucose") is responsible for increased ADCC activity. Moreover, recombinant expression of the enzyme GnTIII in CHO cells permitting the perturbed GlcNAc to be attached to N-glycans can result in the expression of the same antibody expressed in CHO cells that were not transfected by recombination into GnTIII (US 6602684, Umana et al., Nature Biotechnology 17: 176-180 (1999)), which results in an increase in the ADCC activity of certain antibodies. In addition, the knock-out of the gene FUT8, which results in the depletion of core-fucose in CHO cells, can increase the ADCC activity of certain antibodies upon expression in such FUT8 KO CHO cells [US 6946292]. These results demonstrate the importance and complexity of the glycosylation pattern in the activity of the antibody.

However, "exogenous" and hence unoptimized glycosylation patterns are not only detrimental to the activity, but may also be immunogenic. Current expression and production systems for proteins and especially antibodies are mainly rodent-derived cell lines and are based on cell lines from hamsters, mice, or rats, such as CHO, BHK, NSO, Sp2 / 0 and YB2 / 0. Rodent cells are known to be capable of producing a large number of abnormally glycosylated protein products that lack efficacy or are immunogenic under non-optimal conditions. In addition, rodent cells are known to express carbohydrate structures that are significantly different from those expressed in human cells, including the presence of non-human saccharides and the lack of specific human sugar moieties, including, for example, immunogenicity, Less effective, lower yield or sub-structural requirements or folding proteins can be expressed in such cells. Most rodent cells express an alternative N-glycollyl neuraminic acid to N-acetylneuraminic acid ("NeuNAc") that is not present in humans, which are immunogenic and / or immunogenic in humans and / 1-3) Galactose modification ("Gal alpha 1-3 Gal") and / or they lack important carbohydrates such as important alpha 2-6-linked NeuNAc or lack a bisecting GlcNAc. There are also other known or unknown differences. In addition, it is known that glycosylated forms are largely exogenous from rodent production, and that the clone-specific sugar forms can vary widely from clone to CHO, NSO or Sp2 / 0 cells and depend on the mode of production and culture conditions .

Furthermore, the expression system is a human cell, such as a Hek 293 cell or a single human retina derived from human embryonic kidney cells are present for the production of a protein comprising a cellular system PerC6® derived from derived cells, which use recombinant DNA techniques And it was intentionally proliferated. However, although these cell lines are often preferred over non-human cell systems, they still have some disadvantages. They have a unique glycosylation pattern that can be attributed to the glycosylation machinery of individual cells. However, depending on the protein to be produced, they can not carry an optimized glycosylation profile, for example in relation to the activity and / or serum half-life of the protein. In particular, it is difficult to achieve a certain degree and pattern of sialylation with these cells. For example, cell systems known in the art can not provide detectable alpha 2-6 linked sialylation, which is important for serum half-life.

From this fact, it becomes apparent that there is still a need for an expression and production system that can further optimize productivity, homogeneity, and / or antibody activity by providing alternative and / or improved expression systems. Moreover, from this fact, it has been found that certain carbohydrate structures, and in particular human carbohydrate structures, are best for improving the activity or homogeneity of proteins and antibodies, and that cell lines and in particular human cell lines may contain any type of glycosylation pattern to optimize the activity of proteins, It is obvious that it can not be said that it should be provided. Thus, there is a need for an expression system that provides a product having a glycosylation pattern different from that obtained with an expression system known in the art.

The present invention provides a solution to this problem by providing a novel expression system based on human infinite proliferating blood cell lines and especially cells of myeloid leukemia origin. Utilizing infinite proliferating human blood cells is advantageous over systems known in the art since they provide glycosylated profiles that are different from other known human cell systems derived from different tissues (e.g., kidney or retina) . This difference can be beneficial in terms of activity, homogeneity and product yield. Moreover, such cells can be transfected and grown in suspension under serum-free conditions.

Thus, according to a first embodiment, a method of producing a protein composition is provided, which comprises the steps of:

(a) introducing at least one nucleic acid encoding at least a portion of said protein in a host cell that is an infinite proliferating human blood cell; And

(b) culturing the host cell under conditions permitting production of the protein composition; And

(c) separating the protein composition.

This method of using infinite proliferating human blood cells improves the production of proteins and especially antibodies in terms of activity, yield and / or homogeneity. This is surprising because so far, infinitely proliferating human blood cells, and especially myeloid leukemia cells, have not been shown to be suitable for protein production and specifically to induce antibodies with improved properties. Although specific rat leukemia cells were used for the expression of antibodies, the latter may express carbohydrate moieties such as NeuGc and Gal alpha 1-3 Gal, which may be immunogenic in humans, as the glycosylation mechanism between human and rat is critically different . Rat YB2 / 0 leukemia cells have been described to produce antibodies with higher levels of bispecific GlcNAc or higher ADCC activity due to intense down-regulation of core-fucose.

It is even more surprising that the problem could be solved by the present invention using infinite proliferating human blood cells, and particularly myeloid cells, because the cell line K562 - myeloid leukemia cells - was described and suggested by gene hybridization of GnTIII [Yoshimura M et al., Cancer Res. 56 (2): 412-8 (1996)] Expressing the gene FUT8 which expresses the fucosyltransferase causing the addition of the proposed core-fucose by RT-PCR without expressing the enzyme for the bisecting GlcNAc [Example 1]. This is surprising because K562 is resistant to lectin treatment because it is strongly bound by lectin LCA, which is a strongly down-regulated basis for cell-negative or core-fucosylation. Because of this information on the glycosylation profile of K562 cells, it was not expected that such cells could improve the activity of the antibody expressed therein, since it was assumed that there is no glycosylation mechanism necessary for favorable activity patterns. It is also surprising that proteins and especially antibodies having increased binding activity, improved homogeneity and / or higher yield can be produced and achieved by the production methods of the present invention compared to cells of the current expression system.

The strategy of the present invention is to provide a protein system capable of providing a protein product with a post-translational modification as close as possible to a human system, thus achieving a system that can have non-or less immunogenic and / or improved activity in a human system To produce a suitable human cell line. The object is to provide a tailor-made glycosylation pattern for each protein / antibody to be expressed.

Due to the complexity of the carbohydrate structure and the fact that the glycosylation mechanisms of the cells involve hundreds of enzymes involved in their synthesis and that these enzymes are mainly expressed species-specific and tissue-specific, human glycosylation One of the main strategies of the present inventors to achieve is to provide a biotechnologically suitable human expression system that expresses a protein having an optimized glycosylation pattern and in particular an antibody. Because the optimized glycosylation pattern may vary from protein to protein and from antibody to antibody, the present invention provides a cell line that produces a different glycosylation pattern, thereby providing a production cell line that provides an optimized glycosylation pattern for the individual protein / antibody product To be selected.

Accordingly, there is provided a method of producing a protein composition, preferably an antibody molecule composition, having a defined glycosylation pattern, including the steps of:

(a) introducing at least one nucleic acid encoding a protein or at least a portion thereof in an infinite proliferating human blood cell as a host cell;

(b) culturing the host cell under conditions that allow production of the protein composition; And

(c) isolating said protein composition having the intended glycosylation properties.

To obtain a protein composition and especially an antibody composition having improved properties according to the present invention, a host cell is selected to produce a protein / antibody composition having at least one of the following glycosylation properties:

(i) does not contain detectable NeuGc.

As outlined above, NeuGc glycosylation may have immunogenicity in humans. Therefore, it is preferable to avoid each glycosylation as much as possible. Each glycosylation utilizes infinitely proliferating human blood cells and is avoided, particularly using host cells of human myeloid leukemia origin.

(ii) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096] in comparison with the same amount of protein molecules in at least one protein molecule composition of the same protein molecule.

Galactose residues were found primarily as beta 1-4 bound to the GlcNAc residue on the antenna of the complexed N-glycan of the antibody, but also beta-1, 3 binding was also found. However, they are generally produced in a triantennary structure. The effect of the degree of galactosylation on the activity is particularly relevant to the significant antibody. It has been demonstrated that galactose depletion results in reduced CDC activity. Thus, it may be desirable to have a high degree of galactosylation. Galactosylation may also play an important role for other proteins.

When referring to the entire carbohydrate structure of a protein molecule, all galactosylation of the protein molecule is considered. When analyzing the carbohydrate structure at a particular glycosylation site of a protein molecule, the focus is on the carbohydrate structure (s) attached to the specific carbohydrate structure (s), such as Asn 297 of the Fc portion of the antibody molecule (see FIG. When evaluating a particular structure, the type / composition of this specific structure is determined. The entire carbohydrate unit and specific carbohydrate chains could also be referenced to define these characteristics (these are synonyms).

(iii) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096], at least 5% higher in the amount of G2 structure compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule.

In particular, when an antibody is produced according to the method of the present invention, a high amount of G2 structure is advantageous. The "G2 structure" defines the glycosylation pattern, where galactose is found at both ends of the biantennary structure bound to the Fc region in the case of antibodies (see FIG. 18). When a single galactose molecule is found, it is referred to as a G1 structure, and a case where galactose is not present is a G0 structure. G2 glycosylation patterns have often been found to improve the CDC of antibodies. Thus, a higher amount of G2 that exceeds at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 95% or even 100% Structure is present in the resulting protein / antibody composition. Suitable cell lines to achieve individual high G2 glycosylation patterns are described herein.

Since the degree of high total galactosylation is often favorable for the CDC of the antibody, it is preferred that 60%, 65%, 70%, 75%, 70%, or more of the total carbohydrate structure or protein, especially the carbohydrate structure at a particular glycosylation site of the antibody molecule, It is often desirable to obtain G2 and / or G1 structures in excess of 80%, 85%, 90%, 95% or even 95%.

According to a further embodiment, a G2 structure in which more than 35% (40%, 45%, 50%, 55%, 60%, 65% or more than 70%) G2 structure is present in the total carbohydrate structure or in the protein molecules Lt; RTI ID = 0.0 > glycosylation < / RTI >

(iv) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. Has at least a 5% lower amount of G0 structure compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from CRL-9096.

As outlined above, a high degree of galactosylation is generally advantageous. Thus, the cell line is preferably selected to avoid G0 structure. The amount of G0 structure is preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75% %, Less than 90%, or even lower.

According to a further embodiment, a G0 structure of less than 22% (20%, 18%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5% Is present on the carbohydrate structure at one particular glycosylation site of the protein molecule in the protein molecules of the protein molecule composition.

(v) does not contain detectable terminal Gal alpha 1-3Gal.

As outlined above, Gal alpha 1-3 Gal glycosylation may be immunogenic in humans. This glycosylation is characterized by a pattern in which the second galactose residue binds at the alpha 1, 3 position of the first galactose residue resulting in a highly immunogenic Gal alpha 1-3 Gal disaccharide. Endogenously proliferating human blood cells and host cells, particularly of human myeloid leukemia origin, are used to avoid individual adverse glycosylation.

(vi) CHOdhfr- [ATCC No. 2] for expression of the entire carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition. CRL-9096], at least 5% lower than that of the same amount of protein molecule in the at least one protein molecule composition of the same protein molecule.

Fucose residues are found at different sites within the N-glycan tree, in particular as follows:

Alpha 1, 6 bound to a GlcNAc residue adjacent to an amino acid strain;

Alpha < / RTI > 1,3 and alpha 1,4 attached to an antinuclear located at the GlcNAc residue;

- Alpha 1,2 coupled to an antinergy located at the Gal moiety.

On antibodies attached to N-glycans, very large numbers of fucose residues are found with 1,6 linked to adjacent GlcNAc residues (so-called "core fucose"). It has been found that the absence of core fucose on the reducing end of N-glycans attached to the antibody enhances the ADCC activity of the antibody by 25- to 100-fold. Due to this beneficial effect on ADCC, the amount of fucose is much lower than that of CHOdhfr- [ATCC No. 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70% or more of at least one protein molecule composition of the same protein molecule isolated from the same protein molecule, , 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, 1500% or 2000%. The present invention also provides a specially engineered cell line that achieves individual low overall fucosylation.

According to a further embodiment, the host cell comprises a carbohydrate of the entire carbohydrate structure lacking fucose or a carbohydrate of at least one specific carbohydrate structure at the specific glycosylation site of the protein molecule in the protein molecule of the protein molecule composition At least 10% (15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% Is selected to produce a glycoprotein. With regard to the antibody, it is particularly preferred that the N-glycoside-linked carbohydrate chain linked to the Fc region comprises a reducing end comprising GlcNAc, wherein the carbohydrate chain is linked to the 6-position of GlcNAc at the reducing end of the carbohydrate chain It does not contain fucose.

(vii) one or more carbohydrate structures containing a bisecting GlcNAc.

The bisecting N-acetylglucosamine (bisGlcNAc) is often found in beta-1,4 attached to the central mannose residue of the tri-mannosyl core structure of N-glycans found in antibodies. The presence of bisecting GlcNAc in the central mannose residue of the antibody Fc-N-glycan increases the ADCC activity of the antibody.

According to a further embodiment, the host cell is characterized in that the resulting protein contains two equivalents of GlcNAc and, compared to the individual carbohydrate structures which do not contain fucose, Is selected to include more of the carbohydrate structure of the entire carbohydrate unit of the molecule (or of at least one particular carbohydrate chain at a particular glycosylation site of the protein molecule).

According to a further remedy, said host cell is characterized in that the resulting protein comprises a bispecific GlcNAc and a fucose-free individual carbohydrate structure, wherein the resulting protein comprises the bispecific GlcNAc and fucose The carbohydrate structure of the entire carbohydrate unit of the protein molecule (or of at least one particular carbohydrate chain at a particular glycosylation site of the protein molecule).

(viii) Expression of CHOdhfr- [ATCC No. Has a modified sialylation pattern relative to the sialylation pattern of one or more protein molecular compositions of the same protein molecule isolated from < RTI ID = 0.0 > CRL-9096.

The effect of sialylation degree / pattern on activity, half-life and bioavailability varies between different proteins / antibodies. Thus, for each protein / antibody molecule, it is advantageous to first determine the optimized sialylation pattern using the screening method according to the present invention, prior to establishing production with the most suitable host cell that provides the desired glycosylation pattern Do. For example, there are several published documents reporting the negative effects of sialic acid residues present on Fc glycans of downstream effects, i. E. Antibodies to CDC and ADCC. However, it has been found that high sialylation extends the half-life of the sialylated molecule. Thus, depending on the protein / antibody being generated, different sialylation patterns may be advantageous and the present invention may provide proteins / antibodies that exhibit optimized glycosylation patterns by providing infinitely proliferating human blood cell lines with different sialylation activities .

According to one embodiment, the host cell expresses CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule isolated from the same protein molecule (Preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% %, 85%, 90%, > 95%) to produce a protein having a reduced degree of sialylation with a smaller amount of sialic acid. According to one embodiment, the product does not even contain a detectable NeuNAc. Depending on the protein / antibody being generated, the presence of sialic acid and in particular NeuNAc may not contribute to the activity of the protein / antibody. In this case, it may be advantageous to avoid sialic acid glycosylation to produce a more homogenous product. This is because the NeuNAc glycosylation pattern may vary in the resulting protein composition. This can lead to difficulties in management approval of the product since the product is due to the different NeuNAc content, which is less homogeneous.

In the case of proteins / antibodies that are not dependent on the presence of NeuNAc glycosylation in activity, avoidance of NeuNAc glycosylation may be advantageous in increasing homogeneity. However, "undetectable NeuNAc" does not necessarily mean that NeuNAc is not present at all. In other words, embodiments that have somewhat less NeuNAc (e.g., 1 to 10%) are also included. An example of an individual protein is FSH.

According to one embodiment, the product encodes CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% 400%, 450%, > 500%) and a reduced amount of sialylation with a smaller amount of NeuNAc. This embodiment is advantageous where the protein / antibody is expressed, where sialylation is assumed to have a negative effect on the activity of the protein / antibody.

Individual glycosylation (absence or very low levels of sialic acid, particularly NeuANc) can be achieved using sialylated deficient cells such as NM-F9 and NM-D4 in serum-free medium.

According to a further embodiment, the product is characterized by an overall carbohydrate structure or a carbohydrate structure at one particular glycosylation site of the protein molecule in the protein molecule composition, such as CHOdhfr- [ATCC No. At least 15% (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the same amount of the protein molecule in the at least one protein molecule composition of the same protein molecule isolated from CRL- Or more of the NeuNAc in terms of the amount of sialylation that is greater than the amount of neuNAc in the sample (eg,%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%

As outlined above, individual increased degrees of sialylation can provide a positive effect by prolonging the serum half-life of the protein. In this case, CHOdhfr- [ATCC No. Nos. Using a cell line that provides a higher degree of sialylation than that achieved in < RTI ID = 0.0 > CRL-9096] < / RTI > and also provides a greater degree of sialylation than that achieved in sialylated deficient cells (e.g., NM-F9 and NM-D4) , Where the precursor needs to be added to generate sialylation. However, even when individual precursors are added to grow such sialylated deficient cells, the cells generally have about 50 to about < RTI ID = 0.0 > 50% < / RTI > It only reaches a degree of sialylation of 60%. Thus, for embodiments targeting higher degrees of sialylation, it may be desirable to use cell lines that are capable of providing individual degrees of sialylation without using NM-F9 and NM-D4.

According to a further embodiment, the product comprises an alpha 2-6 linked NeuNAc. In addition, there may be some degree of alpha 2-3-linked NeuNAc. With respect to some proteins / antibodies, the presence of NeuNAc glycosylation is particularly advantageous with regard to the half-life of the protein / antibody. It is advantageous to provide an alpha 2-6 linked NeuNAc, since this glycosylation pattern is similar to the human glycosylation pattern. Rodent cells generally provide an alpha 2-3-linked NeuNAc. In addition, other existing human cell lines can not provide sufficient alpha 2-6 linked NeuNAc glycosylation.

Suitable cell lines that provide individual glycosylation patterns are, for example, NM-H9D8 and NM-H9D8-E6.

According to a further embodiment, the expression of CHOdhfr- [ATCC No. In a specific glycosylation site of the protein molecule of the protein molecule composition, as compared to the same amount of the protein molecule in one or more protein molecule compositions of the same protein molecule isolated from the same protein molecule isolated from the same protein molecule, A host cell that produces a protein comprising a carbohydrate chain linked by at least 20% of the charged N-glycosides of the carbohydrate unit is used.

The charge profile of the carbohydrate chain should be considered as it may affect the properties. The chemical group that carries the carbohydrate chain is, for example, sulfur or sialic acid.

According to an alternative embodiment, the product is CHOdhfr- [ATCC No. CRL-9096], at least one specific carbohydrate chain or at least one specific carbohydrate moiety at the specific glycosylation site of the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in the at least one protein molecular composition of the same protein molecule Carbohydrate chains linked by charged N-glycosides that are at least 20% less than the total carbohydrate unit.

According to a further embodiment, the host cell comprises at least 2% (5 < RTI ID = 0.0 > (%) < / RTI > of the total carbohydrate unit or at least one particular carbohydrate chain at the specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing bisecting GlcNAc Wherein the carbohydrate structure comprises a carbohydrate structure of at least 10%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%

According to one embodiment, the host cell is used to produce a protein that exhibits the following characteristics:

- cell line CHOdhfr- [ATCC No. Increased yield and / or improved homogeneity compared to one or more protein molecular compositions of the same protein molecule at the time of expression in the CRL-9096; And / or

- cell line CHOdhfr- [ATCC No. CRL-9096] that is at least 10% higher than the yield of one or more protein molecule compositions from the same protein molecule upon expression in the same protein; And / or

- Improved homogeneity which is improved glycosylation homogeneity, wherein said antibody molecule composition comprises the cell line CHOdhfr- [ CRL-9096], the degree of sialylation is lower than the degree of sialylation of one or more antibody molecule compositions from the same protein molecule; And / or

- If the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. Has a cytotoxicity of increased Fc-mediated cell that is at least 2-fold higher than the cytotoxicity of Fc-mediated cells of one or more antibody molecule compositions from the same antibody molecule upon expression in CRL-9096; And / or

- If the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. CRL-9096] has at least 50% higher antigen-mediated or Fc-mediated binding than the binding of one or more antibody molecule compositions from the same antibody molecule.

As outlined above, individual characteristics can be obtained by optimizing the glycosylation of the proteins disclosed herein. It is also an amazing observation that the binding profile can be altered or improved with some antibodies based on the glycosylated profile. Suitable host cells are also disclosed herein.

According to a further embodiment, the improved homogeneity of the protein molecular composition is an improved glycosylation homogeneity of the protein molecular composition comprising at least one of the following characteristics:

- NeuGc not detectable;

- undetectable NeuNAc;

- cell line CHOdhfr- [ATCC No. CRL-9096] than the protein molecular composition from the same protein molecule NeuNAc;

- detectable alpha 2-6 bound NeuNAc.

According to a further embodiment, the host cell is selected to produce a protein composition comprising a protein molecule having one of the following characteristic glycosylation patterns:

(a) - does not contain detectable NeuGc

- Does not contain detectable Gal alpha 1-3Gal

- a galactosylation pattern as defined in claim 2

- has a fucose content as defined in paragraph 2

- Contains bisecting GlcNAc

- cell line CHOdhfr- [ATCC No. CRL-9096], or an increased amount of sialic acid relative to a sialylated deficient cell line such as NM-F9 and NM-D4 as compared to the protein composition of the same protein molecule.

(b) does not contain detectable NeuGc

- Does not contain detectable Gal alpha 1-3Gal

- a galactosylation pattern as defined in claim 2

- has a fucose content as defined in paragraph 2

- Contains bisecting GlcNAc

- cell line CHOdhfr- [ATCC No. CRL-9096], as compared to the protein composition of the same protein molecule.

(c) - does not contain detectable NeuGc

- Does not contain detectable Gal alpha 1-3Gal

- a galactosylation pattern as defined in claim 2

- has a fucose content as defined in paragraph 2

- Contains bisecting GlcNAc

- Includes 2-6 NeuNAc.

In addition, suitable characteristic combinations that result in improved features are shown in Table 9.

According to the present invention, the term " protein molecule " refers to a protein of interest or its active fragment and / or mutant, whereby any protein, preferably any glycoprotein of human origin, can be used. The term protein molecule refers to any polypeptide molecule or portion thereof. It can be encoded by one or several nucleic acids. It may be produced as a fusion protein with the secreted form or fraction thereof or a fusion partner. Preferably, the protein is secreted into the supernatant. This embodiment is particularly advantageous with respect to the overall production process, for example, since a shedding step (e.g. using a porbole ester) can be avoided.

Examples of mammalian glycoproteins include molecules such as cytokines and their receptors, such as tumor necrosis factors TNF-alpha and TNF-beta; Renin; Human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipoprotein; Alpha-1-antitrypsin; Insulin A-chain and B-chain; Gonadotropic hormones such as FSH, luteinizing hormone (LH), thyroid stimulating hormone and human chorionic gonadotropin (hCG); Calcitonin; Glucagon; Coagulation factors such as Factor VIIIC, Factor IX, Factor VII, Tissue Factor and von Willebrands Factor; Anti-coagulation factors such as protein C; Atrial sodium diuretics; Pulmonary surfactants; Plasminogen activators such as europinase, human urine and tissue-type plasminogen activator; Bombesh Shin; Thrombin; Hematopoietic growth factors; Enkephalinae; Human macrophage inflammatory protein; Serum albumin, such as human serum albumin; Mullerian-inhibiting substances; Relaxacin A-chain and B-chain; Prolylaxin; Mouse gonadotropin-related peptides; Vascular endothelial growth factor; Receptors for hormones or growth factors; Integrin; Proteins A and D; Rheumatoid-like factors; Neurotrophic factors such as bone-derived neurotrophic factor, neurotrophin-3, -4, -5, -6 and nerve growth factor-beta; Platelet-derived growth factor; Fibroblast growth factor; Epidermal growth factor; Convertible growth factors such as TGF-alpha and TGF-beta; Insulin-like growth factor-I and -II; Insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 and CD-19; Erythropoietin (EPO); Bone inducers; Immunotoxins; Bone morphogenic protein; Interferons such as interferon-alpha, -beta, and -gamma; Colony stimulating factors (CSF's) such as M-CSF, GM-CSF and G-CSF; Interleukins (IL's), such as IL-1 through IL-12; Superoxide dismutase; T-cell receptors; Surface membrane protein; Collapse promoting factor; Antibodies and immunoadhesins; Glycophorin A; There is MUC1.

Many of the above-mentioned glycoproteins belong to the class of common hormones occurring in the cells of the immune system, both lymphocaine and monocaine, and the cytokines mentioned herein as others. This definition includes, but is not limited to, hormones that function locally and that do not circulate in the blood, and hormones that, when used in accordance with the present invention, will result in a change in the immune response of an individual. Examples of additional suitable immunomodulating cytokines include interferons (e.g., IFN-alpha, IFN-beta and IFN-gamma), interleukins (e.g., IL-1, IL-2, IL-3, IL- , TNF-alpha and TNF-beta), erythropoietin (EPO), FLT-10, and IL- 3 ligands, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), CD2 and ICAM. When taking erythropoietin, the molecule is thought to cause progenitor cells that mature into red blood cells, while Trombopoietin is thought to induce progenitor cells that follow the platelet pathway. CSF refers to a family of lymphocines that induce progenitor cells found in the bone marrow and differentiate into specific types of mature blood cells. The specific type of mature blood cells resulting from progenitor cells is dependent on the type of CSF present. Similarly, granulocyte-macrophage colony formation is dependent on the presence of GM-CSF. In addition, the cytokines of other mammals that are substantially homologous to IL-2, GM-CSF, TNF-alpha and other human forms would be useful in the present invention if demonstrated to exhibit similar activity in the immune system. Adherent or ancillary molecules or combinations thereof may be applied alone or in combination with cytokines.

Similarly, proteins that are substantially similar to any particular protein but have relatively few changes in the protein sequence will also be used in the present invention. Some minor alterations in the amino acid sequence of a protein sequence may not interfere with the functional capabilities of the protein molecule, so that a protein that functions as a parent protein but somewhat different from the current known sequence may be prepared in the present invention. Thus, individual variants that maintain biological function are also included.

Preferred glycoproteins are selected from the group comprising glycophorin A, EPO, G-CSF, GM-CSF, FSH, hCG, LH,

All of the above-mentioned protein molecules may be fused to other peptide or polypeptide sequences, such as, but not limited to, linkers, activation molecules or toxins.

In a preferred embodiment of the invention, the nucleic acid encodes the secreted form of the protein or fragment thereof. In a preferred embodiment, the secretory form lacks a transmembrane domain.

According to the present invention, the term " protein molecular composition " refers to molecules of any protein molecule expressed in host cells of the invention that are expressed and particularly isolated according to the methods of the present invention. The protein molecular composition comprises one or more protein molecules. The sugar form of the protein molecule refers to a protein molecule having a particular glycosylation or carbohydrate chain that differs in at least one sugar building block and includes, for example, additional galactose, sialic acid, bisecting GlcNAc, fucose, There is another sugar modification such as acetylation or sulfation from another sugar form of the same protein molecule. In another embodiment of the present invention, the protein molecular composition may comprise molecules of more than one protein molecule expressed in the host cell. In a preferred embodiment, the protein molecular composition of the invention comprises at least one of the cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / 0, or PerC.6 or mouse hybridoma, preferably CHOdhfr- [ATCC No. 1]. CRL-9096] of the protein molecule that mediates higher activity than the protein molecular composition of the same protein molecule. In a further preferred embodiment, the protein molecular composition of the present invention comprises a cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / 0, or a PerC.6 or mouse hybridoma, preferably CHOdhfr- [ ATCC No. Lt; / RTI > of a protein molecule that mediates higher activity than the protein molecular composition of the same protein molecule obtained from the same protein molecule obtained from CRL-9096.

According to the present invention, the term " antibody molecule " means a molecule comprising any whole antibody or antibody fragment or antibody fragment . The whole antibody is of animal origin, for example, this limit is but are of human, simian, rodent, mouse, rat, hamster, rabbit, camel, bird, chicken or IgG of shark origin, IgG1, IgG2, IgG3, IgG4, IgM, IgA, Immunoglobulin molecule or any molecule of any class or subtype or any molecule, including one or more immunoglobulin domains known to those of ordinary skill in the art, including, but not limited to, IgD, Including a protein sequence from antibodies of various animal origin, such as, for example, chimeric or humanized antibodies in which various proportions of murine and human sequences are combined and / or mutated into whole antibodies and / Lt; / RTI > In another embodiment of the present invention, the whole antibody may be an entire antibody as described above having one or more additional amino acid or polypeptide sequences.

In a preferred embodiment, the whole antibody is a human, humanized, or chimeric IgG, IgGl, IgG2, IgG3, IgG4, or IgM comprising a human Fc region. In a further preferred embodiment of the invention said whole antibody is human, humanized or chimeric IgGl, IgG4 or IgM carrying a human Fc region. The human Fc region comprises at least 20 amino acid sequences of the constant domain of the Fc region of a human antibody, more preferably at least 100 amino acids, preferably comprising at least one Fc domain of a human antibody, Human C gamma 2 domain, and more preferably all constant domains of the Fc region of a particular class or subclass human antibody. The human Fc region may comprise a human sequence in which one or more amino acids have been mutated.

In a most preferred embodiment of the invention, the whole antibody molecule is generated from (i) a transgenic mouse produced from, for example, blood cells or cells for human antibody production, or a mouse antibody locus at least partially exchanged by a human antibody sequence Or (ii) at least a portion of the variable region of the murine or rat antibody, e. G., One or more amino acids of the framework region or framework, is mutated to include a human constant domain that is humanized or less immunogenic in humans Humanized whole antibody, or (iii) the variable region is a murine or rat and a human constant region.

Methods for constructing, identifying, testing, optimizing, and selecting these antibody molecules, with or without suitable additional sequences, as well as the complete human antibody, humanized whole antibody, and chimeric whole antibody or portions thereof, Methods for constructing, identifying, testing and selecting the most suitable nucleic acid to be used are well known to those skilled in the art .

The antibody fragment is any fragment of an antibody comprising more than 20 amino acids, preferably more than 100 amino acids from the whole antibody. In a preferred embodiment, the antibody fragment is an antibody, such as multibodies, single domain antibodies or affibodies, comprising multiple binding domains such as Fab, F (ab) 2, diabodies, triabodies or tetrabodies As shown in FIG. In another preferred embodiment, the antibody fragment comprises an Fc region having all or part of its constant domain, preferably comprising a second domain (C gamma 2 domain). In another embodiment, the antibody fragment is a truncated whole antibody lacking one or more amino acids, a polypeptide stretch or an entire domain. Such molecules may be combined with additional sequences such as linkers to stabilize or improve the binding of the molecule.

The molecule comprising said antibody fragment is any molecule comprising any of said antibody fragments or other immunoglobulin domains of at least 20 amino acids. In a preferred embodiment, the molecule comprising an antibody fragment is an antibody fragment in which the antibody fragment has a different protein sequence, such as an effector sequence, such as a cytokine, a co-stimulatory factor, a toxin, An antibody fragment from another antibody to produce a molecule having specificity, or a multimerization sequence such as a MBP (mannan binding protein) domain that results in a multimerization of the binding domain, or a tag, such as a detection, A stabilizing sequence, a localization signal or a linker. In another preferred embodiment, the molecule comprising the antibody fragment is a fusion molecule comprising an Fc region of the whole antibody, or a portion thereof, preferably comprising a second constant domain (C gamma 2 domain). The fusion molecule may be introduced into a gene means in which the molecule is encoded by one nucleic acid or fused by co-expression of two or more nucleic acids, thereby causing fusion to occur by non-covalent or covalent protein interaction or by a combination of the two Lt; / RTI > Gene fusion between the antibody fragment and another polypeptide sequence or protein molecule can be achieved by gene engineering treatment wherein both parts are encoded by a single nucleic acid with or without additional amino acids between them. In a further preferred embodiment, the fusion molecule comprises an antibody fragment, e.g., one or more binding regions from a single domain antibody, or a binding sequence not derived from a Fab or antibody, such as a lectin domain, and a second domain (C gamma domain) An Fc region comprising, or a portion thereof. In another preferred embodiment, the fusion molecule comprises IL-2, IL-12, IL-15, GM-CSF, a peptide toxin, or a portion thereof. They can be obtained, for example, by gene means in which the molecules are encoded by a single nucleic acid or fused by co-expression of two or more nucleic acids, whereby the fusion is caused by non-covalent or covalent protein interactions, Fragments, e. G., Single domain antibodies, Fab or Fab linked to a < / RTI >

Methods for constructing, identifying, testing and selecting these antibody molecules, with or without a suitable additional sequence as well as all such antibody molecules or portions thereof, as well as constructing, identifying, testing and selecting the most suitable nucleic acid encoding these antibody molecules Methods are known to those skilled in the art .

In accordance with the present invention, the term " antibody molecule composition " refers to molecules of any antibody molecule that is expressed in a host cell of the invention that can be isolated. The antibody molecule composition comprises one or more antibody molecules. The antibody composition comprises one or more sugar forms of the antibody molecule. The sugar form of the antibody molecule means a protein molecule having at least one sugar building block that has a particular glycosylation or carbohydrate chain that is different, such as additional galactose, sialic acid, bisecting GlcNAc, fucose, But not limited to, another sugar modification such as acetylation or sulfation from another sugar form of the same antibody molecule. In another embodiment of the present invention, the antibody molecule composition may comprise molecules of more than one antibody molecule expressed in a host cell. In a preferred embodiment, the antibody molecule composition of the invention comprises at least one of the cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / 0, or PerC.6 or mouse hybridoma, preferably CHOdhfr- [ATCC No. 1]. CRL-9096], the molecules of said sugar or sugar forms of the antibody molecule that mediate cytotoxicity and / or improved binding of Fc-mediated cells to a greater percentage than antibody molecule compositions of the same antibody molecule . In a further preferred embodiment, the antibody molecule composition of the present invention comprises a cell line CHO, or CHOdhfr-, or BHK, or NS0, or SP2 / 0, or a PerC.6 or mouse hybridoma, preferably CHOdhfr- [ ATCC No. A greater percentage of the molecules of said sugar or sugar forms of the antibody molecule that mediate cytotoxicity and / or improved binding of Fc-mediated cells than antibody molecule compositions of the same antibody molecule obtained from CRL-9096 .

According to the present invention, the term " host cell of human myelogenous leukemia origin " or equivalent expression refers to any cell or cell line of human myeloid leukemia origin, or any human myelogenous or myeloid precursor cell or cell line obtainable from a leukemia patient, Refers to any cell or cell line that can be obtained from a donor, or a cell or cell line derived from any of the above host cells, or a mixture of cells or cell lines comprising at least one of the above-mentioned cells .

In another embodiment of the invention, host cells of human myeloid leukemia origin or endogenous proliferating human hematopoietic cells of the invention are those cells or cell lines obtained by fusing one or more of the above-mentioned host cells, But are not limited to, those of a myeloid leukemogenesis having other cells of animal origin, such as, but not limited to, B cells, CHO cells. Those skilled in the art will recognize and utilize methods for obtaining, producing and / or infinitely multiplying suitable cells and cell lines from suitable sources and humans for suitable host cells of human myelogenous leukemia origin.

The term cell or cell line derived from said host cell can be obtained by any means of culture and cloning, with or without preliminary mutagenesis or gene engineering treatment of said host cell of myeloid leukemia origin, Means any cell or cell line which can be obtained by any means including the selection of a cell or cell line derived from the host cell. The culturing and cloning can be carried out by using a single cell cloning method, such as flow cytometry based on limited dilution or cell sorting, preferably by means of cloning of multiple rounds of cells and cells, ≪ / RTI > and even from cultures of cell clones. In a preferred embodiment, the cell or cell line derived from the host is selected by binding to a lectin or carbohydrate-binding antibody. Such mutations may be carried out by processes known to those skilled in the art as physical, chemical or biological mutagens, such as, but not limited to, spinning, alkylating agents or proteins such as EMS (ethyl methanesulfonate), lectins, or viral particles. Such gene engineering treatments may include, but are not limited to, gene knock-out through site-specific homologous recombination, utilization of transosons, site-specific mutagenesis, transfection of specific nucleic acids or silencing of genes, Can be carried out by methods known to those skilled in the art. Such culturing and cloning methods, mutations and mutagenesis and gene engineering treatments are well known to those skilled in the art, and some embodiments are disclosed in detail in WO2005 / 017130 A2, US2003 / 0115614 A1, or disclosed herein. One skilled in the art can select and / or employ and / or modify a suitable method or combination of methods for producing a suitable cell or cell line derived from the host cell of the invention.

The cell or cell line derived from the host cell may be characterized by the characteristics of those cells which are advantageous compared to their parental cell line or cell line, such as, but not limited to, short double times, faster growth, Can be grown under serum-free conditions and / or in a protein-free medium, higher cloning efficiency, higher transfection efficiency in the case of DNA, higher expression rate of the antibody molecule composition, better expression of the expressed antibody molecule composition Higher activity, higher homogeneity of the expressed antibody molecule composition, and / or stronger magnification. Methods of selecting such cells with favorable properties are known to those skilled in the art or are disclosed herein. (A) incubating human myeloid leukemia cells with an antibody that recognizes a lectin- or desialylated epitope or an epitope lacking sialic acid, but which is not bound or significantly less bound to the sialylated form of the epitope; (b) Isolating cells bound to the lectin or antibody, (c) culturing the isolated cells for a suitable period of time, and (d) contacting cells, cells or cell clones that are strongly bound to an antibody that binds to an epitope carrying a lectin or sialic acid Lt; RTI ID = 0.0 > of the < / RTI > host cell of the invention.

More preferably, the antibody recognizing the epitope lacking the lectin or desialylated epitope or sialic acid is Nemod-TF1, Nemod-TF2, A78-G / A7, or PNA and the human myeloid leukemia cell is a cell line K562 , KG-1, MUTZ-3, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-H9, H9 and NM-H10.

Methods of producing host cells with favorable biotechnological properties such as high sialylation and rapid cell growth include (i) contacting the human myelogenous leukemia cells of the invention, preferably K562, as a source cell, preferably with K562, Nemod-TF1, Nemod-TF2, A78-A, or Nemod-TF2, which recognize epitopes lacking a lectin or preferably a desialylated epitope or sialic acid but which do not bind or bind less to the sialylated form of the epitope, (Ii) isolating cells bound to the lectin or antibody, (iii) isolating the cells for a suitable period of time, and (iv) an antibody or lectin bound to an epitope that lacks sialic acid, preferably after single cell cloning, such as SNA (Sambucinigra Jipso) or MAL (comprising maak Escherichia no alkylene cis lectin), MAL I (Escherichia maak no alkylene cis lectin I), preferably the selection of a stronger binding to SNA cells, or cell clones.

In a preferred embodiment, the present invention provides a method for the detection of a cell (i) incubating K562 with Nemod-TF1, Nemod-TF2, A78-G / A7, or PNA coupled to magnetic beads, (Iii) culturing the isolated cells for a suitable period of time of about 1 to 6 weeks, and (iv) selecting a cell clone that is tightly bound to the SNA after single cell cloning, Lt; RTI ID = 0.0 > biochemical < / RTI > properties such as growth. In a preferred embodiment, such cells are more tightly bound to SNA than the origin cells, and in another preferred embodiment they grow faster than the origin cells.

In a preferred embodiment of the invention, the progenitor cells are treated with ethyl methanesulfonate prior to step (i).

Those skilled in the art will be able to select the appropriate conditions and methods and optimize them to produce such host cells in view of the present invention. A more detailed description of some steps can be found in WO2005 / 017130 A2 and WO2005 / 080585 A1.

According to one embodiment, infinitely proliferating human blood cells selected from the following Groups 1 to 4 are used as host cells:

(a) a group 1 comprising a host cell having a high sialylation activity such as K562;

(b) sialylation that is low or absent due to genetic defects or expression suppression means (e. g., RNAi); Group 2 comprising a host cell having an activity comparable to and comprising NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] and GTX-2;

(c) a group 3 comprising a host cell having a higher degree of silylation than K562, such as NM-H9 and NM-H9D8;

(d) a group comprising host cells having low or even non-existent fucosylating activity such as NM-H9D8-E6 and NM H9D8-E6Q12.

In a preferred embodiment, the cell line formed is NM-H9D8. NM-H9D8 was selected as the most preferred cell clone formed by the method described above, and this was cloned by Glycotope GmbH, Roberts-Rohrle-Strasse 10, Berlin, Germany 13125, DSMZ-Deutsche Jungung Phone Microorganism < / RTI > and Zellkulturen GmbH ", which was deposited with DSM ACC 2806 on September 15, 2006. Other cell clones such as NM-E-2F9, NM-C-2F5, or NM-H9D8-E6 (DSM ACC 2807) were incubated with one or more lectins and cell sorting by flow cytometry To select by single cell cloning, thereby performing a positive selection process or a combined selection process of negative and positive. To obtain cell clones with stable characteristics, the obtained cell clones were recloned at least once by the limited dilution described above.

In a preferred embodiment of the present invention, the infinitely proliferating human blood cells, preferably host cells of myeloid leukemia origin, are grown and the protein / antibody molecule composition of the present invention is produced under serum free conditions. In a further preferred embodiment, said infinite proliferating human blood cells, preferably host cells of myeloid leukemia origin, are grown under serum free conditions. In addition, nucleic acids encoding protein / antibody molecules can be introduced into these cells and protein / antibody molecule compositions can also be isolated under serum free conditions. Being able to work under serum-free conditions is particularly important when preparing therapeutic proteins because serum contamination is not acceptable in controlled methods.

In a preferred embodiment of the present invention, the host cell of the human myeloid leukemia origin of the present invention is selected from the group consisting of K562, KG1, MUTZ-2, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] A cell or cell line derived from any, a cell or a mixture of cell lines comprising at least one of the above-mentioned cells. The host cells are preferably selected from the group consisting of NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM- E6 DSM ACC 2807, or NM H9D8-E6Q12 [DSM ACC 2856], GT-2X (DSMZ-Deutsche Jung, Germany, Braunschweig, Germany, by Glikotopegembeck, Roberto- ≪ / RTI > deposited with the Von Mikro Organism Ment Challenger Turgen < RTI ID = 0.0 > GmbH < / RTI > on September 7, 2007, DSM ACC), or cells or cell lines derived from any of these cell lines.

Cell NM-F9 [DSM ACC2606] and NM-D4 [DSM ACC2605] were purchased from Nemodai Biosciences, Inc., 13125 Berlin-Rostel-Strasse 10, Berlin, Germany, entitled " Deposited Biological Substances "Lt; RTI ID = 0.0 > Nemod Biotherapeutics < / RTI > GmbH &

In a further preferred embodiment of the invention, the host cells from human myeloid leukemia of the invention are selected from the group consisting of cell or cell line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] Lt; RTI ID = 0.0 > and / or < / RTI >

In a preferred embodiment of the invention, the host cell of the human myelogenous leukemia origin of the invention is a cell or cell line K562 [ATCC CCL-243], or a cell or cell line derived from said host cell.

In a further preferred embodiment of the invention, the host cell of the human myeloid leukemia origin of the invention is a cell or cell line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- Or NM-H9D8-E6, or NM-H9D8-E6, or NM-H9D8-E6, or GT-2X or NM H9D8-E6Q12 or cells or cell lines derived from any of the above host cells.

In an even more preferred embodiment of the invention, the host cell of the human myeloid leukemia origin of the invention is a cell, cell or cell line K562, NM-F9 [ DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM H9D8-E6, GT-2X, or NM M9D8- Are cells, cells or cell lines described below that grow under serum-free conditions, nucleic acids encoding the antibody molecules can be introduced into these cells, and the antibody molecule composition is isolated under serum-free conditions.

In a most preferred embodiment of the present invention, the host cell of the human myeloid leukemia origin of the invention is a cell, cell or cell line NM-F9 [DSM ACC2606] which grows under serum free conditions to produce the antibody molecule composition of the present invention, , NM-D4 [DSM ACC 2605], NM-E-2F9, NM-C-2F5, NM-H9D8, GT-2X, or NM H9D8-E6, or NM M9D8- The nucleic acid encoding the antibody molecule can be introduced into these cells, and the antibody molecule composition is isolated under serum-free conditions.

According to one embodiment, the host cells of infinitely proliferating human blood cells and human myelogenous leukemia origin are selected from the group consisting of sialylated deficient cell lines NM-F9 and NM-D4 or cells or cell lines derived from any of the host cells having the same characteristics It is not. This embodiment is advantageous when it is aimed at high sialylation degree. For these embodiments, the host cell is preferably selected from the group consisting of K562, NM H9D8, NM H9D8-E6, NM H9D8-E6Q12, and host cells derived from any of these host cells.

The cell line described with the present invention has a doubling time of 14 to 24 hours, which depends on the cell line of the present invention, but is very fast compared to other mammalian expression systems.

In addition, no generally suitable high expression vector system is known for high yield of antibody expression in human cell lines, since human cells generally carry the DHFR gene and thus do not allow the use of the dhfr / methotrexate amplification system Because. Thus, according to a further embodiment of the present invention, embodiments are provided that can increase the product yield.

According to this embodiment, a nucleic acid encoding an additional antifolate resistant DHFR-variant is introduced into the host cell. Dihydrofolate reductase, or DHFR, reduces dihydrofolic acid to tetrahydrophosphoric acid, which can be converted to the type of tetrahydrofolate cofactor in 1-carbon transfer chemistry, using NADPH as the electron donor . Antifolates inhibit DHFR enzymes that cause cell death. To provide a nucleic acid encoding an antifolate-resistant DHFR variant, a tool for selecting a nucleic acid-transfected cell is provided, and the nucleic acid to be expressed in the host cell can be amplified by the tool.

The nucleic acid encoding the antifolate-resistant DHFR-variant can be introduced, for example, via a vector separate from the nucleic acid encoding the protein / antibody to be expressed in the host cell. It is preferred that the vector encoding the antifolate resistant DHFR-variant is co-transfected with a vector containing hexane encoding essentially the protein / antibody. In this embodiment, the nucleic acid encoding the protein / antibody to be expressed is in the same gene position as the nucleic acid encoding the antifolate-resistant DHFR-variant, which is advantageous when amplification of the nucleic acid encoding the protein / antibody is desired Lt; RTI ID = 0.0 > genomic < / RTI >

Alternatively, a vector system comprising a nucleic acid encoding at least a portion of said protein to be expressed and a nucleic acid encoding an antifolate resistant DHFR-variant may be used.

The host cells are then incubated with the antifolate. This has the effect that such host cells successfully transfected using the nucleic acid encoding the antifolate resistant DHFR-variant can grow despite the presence of the antifolate. Whereby successfully transfected cells can be selected.

According to a further embodiment, the nucleic acid sequence encoding at least part of said protein / antibody is amplified by stepwise increasing the antipolate concentration in the culture. Increasing concentrations of antifolates in the medium increase the copies of antifolate-resistant DHFR-variants in the genome. It is presumed that this is caused by a recombination event in the cell. Thereby, the number of copies of the nucleic acid encoding at least a portion of the protein to be expressed is also increased when the nucleic acid encoding the protein is located near the antifolate-resistant DHFR variant in the genome, Or by using a vector containing both nucleic acid sequences. By such a mechanism, a host cell expressing the protein / antibody at a higher yield is obtained.

Preferably, the antifolate is methotrexate.

The nucleic acid sequence encoding the protein, preferably an antibody molecule or a portion thereof, is preferably amplified by incubating the host cell with at least two consecutive rounds of antifolate, preferably methotrexate, The concentration of folate, preferably methotrexate, increases by at least 100% in each successive round.

Suitable nucleic acids for providing the antifolate resistant DHFR-variants encode the group of SEQ ID NOS: 1-9, preferably the polypeptide of SEQ ID NO: 1.

Additional details and specific examples of such an amplification system are also described in further detail below.

The present invention also provides a method of producing a protein, preferably an antibody molecule composition,

(a) at least one nucleic acid encoding a protein and preferably an antibody molecule or at least one portion thereof in a host cell of human myelogenous leukemia origin, and a group of SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: Introducing at least one nucleic acid comprising at least one nucleic acid sequence encoding at least one polypeptide of SEQ ID NO:

(b) incubating the host cell with methotrexate, preferably by incubating the host cell with at least two consecutive rounds of methotrexate, preferably at least two consecutive rounds of methotrexate, To thereby increase the concentration of methotrexate in each successive round, preferably by at least about 50%, more preferably by at least about 100%;

(c) culturing the host cell under conditions that allow the protein, preferably an antibody molecule composition, to be produced; And

(d) isolating said protein, preferably an antibody molecule composition.

The present invention also relates to a method of producing a protein composition, preferably an antibody molecule composition, having increased activity and / or increased yield and / or improved homology,

(a) introducing at least one nucleic acid encoding a protein, preferably an antibody molecule or at least a portion thereof, into a host cell of human myelogenous leukemia origin;

(b) culturing the host cell under conditions that can produce the protein composition, preferably an antibody molecule composition; And

(c) isolating the protein composition, preferably an antibody molecule composition, with increased activity and / or increased yield and / or improved homology.

Moreover, the present invention provides a method of producing a protein composition, preferably an antibody molecule composition, having increased activity and / or increased yield and / or improved homology,

(a) at least one nucleic acid encoding a protein, preferably an antibody molecule or at least one portion thereof, in a host cell of human myeloid leukemia origin, and a group of SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: Introducing at least one nucleic acid comprising at least one nucleic acid sequence encoding at least one polypeptide of SEQ ID NO:

(b) amplifying the nucleic acid sequence encoding the antibody molecule or at least a portion thereof by incubating the host cell with methotrexate, preferably by incubating the host cell with at least two consecutive rounds of methotrexate , Thereby increasing the concentration of methotrexate in each successive round, preferably by at least about 50%, more preferably by at least about 100%;

(c) culturing the host cell under conditions that can produce the protein composition, preferably an antibody molecule composition; And

(d) isolating the protein composition, preferably an antibody molecule composition, with increased activity and / or increased yield and / or improved homology.

The nucleic acid encoding said antibody molecule or at least one portion thereof and at least one nucleic acid sequence encoding at least one polypeptide of SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: 1, One nucleic acid or two separate nucleic acids.

In order to select a host cell suitable for obtaining a protein having an optimized glycosylated profile, it is advantageous to carry out the screening / selection method according to the present invention. After a suitable host cell has been determined by the selection method according to the invention, the host cell is then used to produce a protein as defined in claims 1 to 20.

Accordingly, the present invention also provides a method of selecting a host cell for producing a protein having at least one of the following glycosylation characteristics (i) to (viii)

(a) introducing at least one nucleic acid encoding a protein or at least a portion thereof as a host cell into at least two different infarcted human blood cells;

(b) culturing the at least two different host cells, wherein each different host cell produces a protein composition having a glycosylation pattern that is different from the glycosylation pattern produced by the other host cell;

(c) isolating said expressed protein composition having a glycosylation pattern different from at least two different host cells; And

(d) selecting said host cell to produce a protein composition having at least one of the glycosylation characteristics as defined in (i) to (viii) below:

(i) the protein does not contain any detectable NeuGc;

(ii) CHOdhfr- [ATCC No. 2] when the protein is expressed in its entirety in a carbohydrate structure or on a specific glycosylation site of a protein molecule in the protein molecule composition on the carbohydrate structure. Having an increased degree of galactosylation compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CRL-9096;

(iii) CHOdhfr- [ATCC No. 2] when the protein is expressed in its entirety in a carbohydrate structure or on a specific glycosylation site of the protein molecule in the protein molecule composition on its carbohydrate structure. / RTI > more than 5% more G2 structure compared to the same amount of protein molecule in the at least one protein molecule composition of the same protein molecule isolated from CRL-9096;

(iv) CHOdhfr- [ATCC No. 2] when the protein is expressed in its entirety in a carbohydrate structure or on a specific glycosylation site of the protein molecule in the protein molecule composition on its carbohydrate structure. Having an amount of G0 structure of at least 5% less than the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from CRL-9096;

(v) the protein does not contain any detectable terminal Gal alpha-3Gal;

(vi) CHOdhfr- [ATCC No. 2] when the protein is expressed in its entirety in a carbohydrate structure or on a specific glycosylation site of the protein molecule in the protein molecule composition on the carbohydrate structure. / RTI > contains less than 5% less fucose in comparison to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from the same protein;

(vii) comprises at least one carbohydrate structure containing GlcNAc in which the protein is bisected; and / or;

(viii) CHOdhfr- < / RTI > Has a modified sialylation pattern compared to the sialylation pattern of at least one protein molecule composition of the same protein molecule isolated from < RTI ID = 0.0 > CRL-9096.

Details of the glycosylation pattern and cell lines suitable for obtaining such a pattern have been described above and are also applicable and suitable for screening methods for selecting suitable host cells according to the present invention.

It is advantageous to perform each screening step prior to establishing the production process as claimed in claims 1 to 20, since the protein / antibody molecule composition having an optimized glycosylation pattern is produced by this embodiment Since the most suitable host cell can be selected. According to the basic idea of this screening system, proteins of interest are expressed in at least two different cell lines with different glycosylation patterns. For example, one cell line may exhibit high sialylation and the other may exhibit low sialylation (or fucosylation and / or galactosylation) or even unknown glycosylation characteristics. Thus, the products obtained from different cell lines possess glycosylation pattern characteristics for each cell line.

For example, properties of proteins produced in different cell lines can be subsequently measured for activity (eg, ADCC and CDC in antibodies), affinity, serum half-life, and other important characteristics. As a result, it becomes possible to select cell lines with the best glycosylation machinery to obtain proteins optimized for the glycosylation pattern. This method is particularly advantageous because the critical properties (e. G., Affinity, serum half-life, etc.) vary.

Preferably, the protein to be produced exhibits at least one of the following characteristics:

(a) when the protein is an antibody molecule composition, it is a cell line CHOdhfr- [ATCC No. 1]. CRL-9096], which has more than two-fold greater Fc-mediated cytotoxicity than the Fc-mediated cytotoxicity of at least one antibody molecule composition from the same antibody molecule;

(b) when the protein is an antibody molecule composition, it is a cell line CHOdhfr- [ATCC No. 2]. Has an increased antigen mediated or Fc-mediated binding greater than 50% greater than the binding of at least one antibody molecule composition from the same antibody molecule when expressed in a cell line, e.g., CRL-9096;

(c) The protein is expressed in the cell line CHOdhfr- [ATCC No. Has an increased average or maximum yield of the protein molecular composition that is at least 10% greater than the yield of at least one antibody molecule composition from the same protein molecule when expressed in CRL-9096.

According to one embodiment, at least one of the host cells is an infinitely proliferating human blood cell and preferably K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- 2F, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, or cells or cell lines derived therefrom.

At least one host cell derived from human myeloid leukemia may be selected from one of the following groups 1 to 4:

(a) Group 1 comprising a host cell having a high sialylation activity, such as K562 or a cell or cell line derived therefrom,

(b) a cell or cell line derived from NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] and GTX-2 or from them due to genetic defects or expression suppression means Group 2 comprising host cells with non-existent sialylation;

(c) a group 3 comprising a host cell having a higher degree of sialylation than K562, such as NM-H9 and NM-H9D8, or a cell or cell line derived therefrom;

(d) a group comprising a host cell having a low or non-existent fucosylating activity such as NM-H9D8-E6 or a cell or cell line derived therefrom.

Preferably, two or more host cells used in the screening process are selected from the group. However, other screening systems according to the present invention may also include other host cells (e. G., Hek 293 cells) from other sources to further expand the different glycosylation patterns analyzed.

Preferably, the resulting host cell produces an antibody that exhibits one or more of the proteins, in particular the glycosylation patterns shown in Table 9.

In accordance with the present invention, the term " nucleic acid " refers to a nucleic acid that introduces a nucleic acid , one nucleic acid, or two or more nucleic acids, including, but not limited to, electroporation, cationic lipids, calcium phosphate, transfection with DEAE-dextran, &Quot; means any method known to those skilled in the art for introducing into a host cell or cells of a mammal, such as by infection with a viral particle, such as an adenovirus or retrovirus or combinations thereof. One skilled in the art can select and optimize methods suitable for introducing one or more nucleic acids of the invention.

According to the present invention, the nucleic acid encoding the antibody molecule is any nucleic acid that encodes the antibody molecule or at least a portion thereof. Whereby the antibody molecule of the present invention can be encoded by one or more nucleic acid molecules.

The portion of the antibody molecule encoded by the nucleic acid comprises at least one 20 amino acid sequence, more preferably an antibody molecule or at least 100 amino acids of the constant and / or variable domains of the antibody molecule. The included sequences encoding the antibody molecule or at least one portion thereof can be separated by at least one other sequence, such as an intron. The sequence of the domain may comprise at least one amino acid mutation.

According to the present invention, a nucleic acid encoding a protein molecule is any nucleic acid that encodes a protein molecule or at least a portion thereof. Whereby the protein molecules of the present invention can be encoded by single or multiple nucleic acid molecules. Sequences encoding protein molecules or at least a portion thereof can be separated by at least one other sequence, such as, for example, an intron. The sequence of the protein molecule may comprise at least one amino acid mutation.

In a preferred embodiment, the nucleic acid encoding the antibody molecule or at least one portion thereof comprises at least one variable domain and / or at least one constant domain of the antibody molecule, more preferably both domains, Those comprising human sequences in at least a portion of the domains, and even more preferably those comprising the human C gamma 2 domain, and most preferably all of the constant regions of the Fc region of a human class of a particular class or subclass Domain < / RTI > and a variable domain.

According to one embodiment, the protein and in particular the antibody molecule are encoded by a single nucleic acid. In another preferred embodiment, the protein, in particular the antibody molecule, is encoded by two nucleic acids or three nucleic acids.

In a further preferred embodiment, one nucleic acid encodes a portion of an antibody molecule that encodes a variable and / or constant domain of a light chain, the other nucleic acid encodes a variant of a heavy chain and / or encodes at least one constant domain Encoding another portion of the antibody molecule.

According to the present invention, a nucleic acid comprising at least one nucleic acid sequence encoding at least one polypeptide of the group of SEQ ID NO: 1 to SEQ ID NO: 9 is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, Means encoding at least one polypeptide of SEQ ID NO: 1. Any number of these sequences is suitable as long as the sequence can be successfully introduced into the host cell of the invention. In a preferred embodiment, the nucleic acid encodes one, two or three polypeptides of the group of SEQ ID NO: 1 to SEQ ID NO: 9, more preferably one polypeptide, and most preferably SEQ ID NO: 1. In terms of the present invention, the nucleic acid can also encode a polypeptide of SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: 1, which has at least one amino acid mutation, The nucleic acid encoding the antibody molecule or at least a portion thereof should be able to be amplified by the methotrexate described elsewhere.

A nucleic acid sequence encoding a group of SEQ ID NOs: 1 to 9, preferably at least one polypeptide of SEQ ID NO: 1 is part of the same nucleic acid molecule as the nucleic acid encoding the antibody molecule or at least one portion thereof described elsewhere herein Or may be on separate nucleic acid molecules.

In another preferred embodiment of the present invention, a separate nucleic acid comprising a nucleic acid sequence encoding a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9, most preferably SEQ ID NO: 1, Can be introduced into the host cell of the present invention independently from the nucleic acid or nucleic acid encoding it. This may be accomplished by introducing the separate nucleic acids comprising a nucleic acid sequence encoding a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9 before or after introducing the nucleic acid or nucleic acid encoding the antibody molecule of the invention or a portion thereof And the like. In a preferred embodiment this is carried out side by side and in another preferred embodiment the host cell of the invention comprises a nucleic acid sequence which already encodes a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 9, most preferably SEQ ID NO: And the separate nucleic acids.

Further preferred embodiments are described in the Examples.

Amplification of the multiple copies of the nucleic acid encoding the stably incorporated nucleic acid, or preferably the antibody molecule or protein fraction thereof, can be performed elsewhere herein and using methotrexate as described in the Examples.

In a preferred embodiment, the nucleic acid encoding the antibody molecule or a protein that is part of at least one of the above is preferably selected from at least one other genetic element that permits the selection of such cells into which the nucleic acid has been successfully introduced, Resistant genes, such as, but not limited to, genetic elements that are encoded for puromycin or neomycin resistance. These nucleic acids may also contain one or a plurality of gene elements, such as promoters, enhancers, polyadenylation sites, and / or introns, for expression of antibody molecules in the host cells of the present invention; And genetic elements such as the bacterial origin of replication, promoters and elements for selecting transfected bacteria, and antibiotic resistance genes for propagating nucleic acid in bacteria.

Suitable promoters include the promoter of IE (immediate early) gene of cytomegalovirus (CMV), SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SR alpha promoter, EF -1 alpha promoter, and the like. An enhancer of the IE gene of human CMV can be used with the promoter.

These and additional genetic elements are known to those skilled in the art and are selected, combined, optimized, and then introduced into the nucleic acid encoding a protein that is preferably an antibody molecule or at least one part thereof, by one skilled in the art. Preferred genetic elements for preferred embodiments and uses and combinations of said nucleic acids or nucleic acid combinations which preferably encode antibody molecules or proteins that are part of at least one of them are described herein and in the Examples, , Which may be further optimized by a combination of those skilled in the art.

One skilled in the art can construct a nucleic acid vector or element that is harmonized to select and / or combine suitable genetic elements and to introduce one or more nucleic acids of the present invention into a cell line according to the present invention.

A combination of said nucleic acid or nucleic acid encoding a protein which is preferably an antibody molecule or at least one part thereof; The additional gene elements; Introducing them into host cells of the invention for expression of antibody molecule compositions, such as by transfecting them into bacteria for propagation; Methods of constructing, identifying, testing, optimizing, selecting and combining these nucleic acids or nucleic acids and combining them with additional sequences suitable for the selection of such cells to be successfully transfected; And methods for constructing, identifying, testing and selecting the most suitable nucleic acid encoding these antibody molecules are known to those skilled in the art.

The preferred For example, In the example  Describe in detail.

In a preferred embodiment of the present invention, preferably the nucleic acid encoding the antibody molecule or a protein that is at least a portion thereof is not limited to a gene element for selecting the host cell of the invention to which the nucleic acid has been successfully introduced, Or more preferably a promoter such as EF-1 alpha or CMV promoter, more preferably a CMV enhancer, and more preferably, a promoter such as EF-1 alpha or CMV promoter, And a gene element that allows selection of the bacterium transfected with the nucleic acid, such as the ColEl origin of replication for multiplication of said nucleic acid in bacteria, and even more preferably a gene for the increase of said nucleic acid in COS cells Element, e.g., SV40 origin. Such elements are well known to those skilled in the art and may be selected, combined, optimized and utilized by those skilled in the art.

Even in a further preferred embodiment of the invention, the disclosed nucleic acid encoding a protein molecule or at least a portion thereof comprises a single nucleic acid sequence. Preferably, the nucleic acid encoding the protein molecule or at least a portion thereof comprises a gene element encoding a furomycin resistance or neomycin resistance, or one or more sequences selected from the group of SEQ ID NOS: 1 to 9, more preferably SEQ ID NO: 1 Encoding the encoding nucleic acid sequence.

In a further preferred embodiment of the present invention, the disclosed nucleic acid encoding an antibody molecule or at least a portion thereof comprises one or more sequences encoding one or more constant domains and variable domains of the heavy and / or light chain of the antibody molecule.

Even in a further preferred embodiment of the invention, the disclosed nucleic acid encoding the antibody molecule or at least a portion thereof encodes a variable domain and constant domain of the light chain of the whole antibody molecule or a variable domain of the heavy chain of the whole antibody molecule, ≪ / RTI >

In a further preferred embodiment of the present invention, one of the disclosed nucleic acids encodes at least a portion of an antibody molecule comprising one or more sequences encoding variable domains and constant domains of the light chain of the antibody molecule, and the second of the disclosed nucleic acids Encodes at least another portion of an antibody molecule comprising one or more sequences encoding a variable domain of the heavy chain of the antibody molecule and one or more constant domains, preferably all constant domains, both of which are present in the same host cell of the invention . Preferably, one of the nucleic acids further encodes a gene element encoding a furomycin resistance and the other encoding further encodes a gene element encoding neomycin resistance.

Even in a further preferred embodiment of the present invention, the disclosed nucleic acid encoding a protein molecule or at least a portion thereof comprises at least two nucleic acid sequences encoding two amino acid sequences of a protein molecule or at least a portion thereof.

Preferably, one of the nucleic acids further encodes a gene element encoding a furomycin resistance, and one further encoding the gene element encoding one of the other nucleic acids encodes neomycin resistance. More preferably, one of the nucleic acids further encodes a gene element encoding a furomycin or neomycin resistance, preferably a furomycin resistance, and one other nucleic acid is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 9 1, and most preferably the nucleic acid sequence encoding SEQ ID NO: 1.

In a further preferred embodiment of the present invention, the disclosed nucleic acid encoding a protein molecule or at least a portion thereof comprises three nucleic acid sequences encoding three amino acid sequences of a protein molecule or at least a portion thereof.

Preferably, one of the nucleic acids further encodes a gene element encoding a furomycin resistance, wherein one other nucleic acid further encodes a gene element encoding neomycin resistance, wherein one other nucleic acid sequence 1 to SEQ ID NO: 9, most preferably SEQ ID NO: 1.

In a more preferred embodiment of the invention, one of the nucleic acids further encodes a gene element encoding a furomycin or neomycin resistance and the other nucleic acid encodes one or more sequences selected from the group of SEQ ID NO: 1 to SEQ ID NO: 9, Preferably further comprises a nucleic acid sequence encoding SEQ ID NO: 1. In an even more preferred embodiment, one of the nucleic acids comprises a sequence encoding a variable domain and an invariant domain of a light chain and encodes one or more sequences selected from the group of SEQ ID NO: 1 to SEQ ID NO: 9, most preferably SEQ ID NO: Wherein the other nucleic acid comprises a sequence encoding a variable domain of the heavy chain of the antibody molecule and one or more constant domains, preferably all constant domains, and is selected from the group consisting of furomycin or neomycin resistant, Lt; RTI ID = 0.0 > a < / RTI > nucleic acid sequence.

In a further preferred embodiment of the invention the nucleic acid encoding the antibody molecule or at least a portion thereof comprises a variable domain of the heavy chain of the antibody molecule and one or more sequences encoding one or more constant domains and constant domains of the light chain of the antibody molecule and one or more constant Domain, preferably one or more sequences encoding all constant domains.

In a preferred embodiment of the invention, the constant domain encoded by the nucleic acid disclosed above or elsewhere herein is a human constant domain of IgG or IgM, preferably human IgGl, IgG4 or IgM, or one of these domains or Sequences derived from genomic or genomic sequences, preferably comprising one or more introns, are used, which can be selected, constructed and optimized by those skilled in the art.

In a further preferred embodiment of the invention the nucleic acid encoding the antibody molecule of the invention or at least a portion thereof is a EF-1 alpha promoter / CMV enhancer or CMV promoter-derived promoter, preferably a CMV- E < / RTI >

In a further preferred embodiment of the invention, the nucleic acid encoding the antibody molecule of the invention or at least a portion thereof is further comprised of a secretion signal peptide, preferably a T cell receptor secretion signal.

Even in a further preferred embodiment of the invention, the nucleic acid encoding the antibody molecule of the invention, or at least a portion thereof, further comprises a secretion signal peptide, preferably SEQ ID NO: 10.

Those additional elements, as well as combinations of the nucleic acids or nucleic acids encoding the antibody molecules of the invention or proteins that are at least part of the same, and methods of introducing them are well known to those skilled in the art, Methods of identifying, identifying, testing, optimizing, selecting and combining these, and combining them with additional sequences suitable for selection of such successfully transfected cells, and constructing, identifying, testing and selecting the most suitable nucleic acid encoding such antibody molecule Methods are known to those skilled in the art .

The preferred For example, In the example  Describe in detail.

The introduction of the nucleic acid may be transient or stable.

According to the present invention, the term " amplifying a nucleic acid sequence encoding a protein or antibody molecule or at least a part thereof by culturing the host cell with an antifolate, in particular methotrexate, " refers to the expression of a protein, One or more nucleic acids encoding one or more nucleic acids that encode an antifolate-resistant DHFR variant, preferably one or more nucleic acids of SEQ ID NOS: 1 to 9, preferably one of SEQ ID NO: 1 Means that the host cells of the invention disclosed herein in which the above polypeptides are introduced are cultured by one or more antifolates, preferably methotrexate. Typical incubation times are from one week to two weeks at a conventional concentration of about 20 nM to 3000 nM, preferably about 50 nM to 2000 nM, more preferably about 100 nM to 2000 nM for each round. The duration of the antifolate / methotrexate amplification treatment and the concentration of the nucleic acid of the present invention and the vector thereof can be optimized by those skilled in the art and are disclosed in preferred embodiments of the examples. Optimal amplification conditions may be different between the use of the different protein / antibody molecules being encoded and the different nucleic acid or nucleic acid constructs disclosed above, or combinations thereof. Those skilled in the art will be able to select and optimize the most suitable conditions and nucleic acids of the present invention. Said amplification is particularly advantageous in the case of cultures without antifolate / methotrexate or with a nucleic acid sequence encoding one or more antifolate resistant DHFR variants, particularly when the protein / antibody < RTI ID = 0.0 > Resulting in the integration of more nucleic acid radiations encoding the molecule or at least a portion thereof into the genome of the host cell and / or resulting in increased production of the protein / antibody molecule composition.

In a preferred embodiment of the invention, as described elsewhere herein, including preferred embodiments, one or more nucleic acid sequences encoding a group of SEQ ID NO: 1 to SEQ ID NO: 9, preferably one or more polypeptides of SEQ ID NO: 1, The host cell for amplifying a nucleic acid or nucleic acid encoding a protein / antibody molecule or at least a portion thereof, introduced into the host cell into which the one or more nucleic acids is incorporated, is a human myelocytic leukemia origin , preferably a cell or cell line NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6, NM-D9 [DSM ACC2606], NM-E-2F9, NM- NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9 or NM-E-2F9, or a cell or a cell line derived from any of the aforementioned host cells, NM-C-2F5, NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X NM-E-2F9, NM-C-2F5 < / RTI > or NM-C-2F9, or a cell or cell line derived from any of the above host cells, and even more preferably a cell or cell line NM-F9 [DSM ACC2606] , NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or the host cell.

In a preferred embodiment of the invention, the host cell is cultured with at least two consecutive rounds of anti-folate / methotrexate so that the concentration of antifolate / methotrexate is preferably at least about 50%, more preferably at least about 50% Is increased to about 100% or more. Even in a further preferred embodiment of the invention, the host cell is treated with at least 3, more preferably 4, more preferably 5, and even more preferably 6 consecutive rounds of anti-folate / methotrexate By culturing, the concentration of antifolate / methotrexate is preferably increased by at least about 50%, more preferably by about 100% or more, in each successive round. More preferred is a concentration of antifolate / methotrexate of about 20 nM to 3000 nM, more preferably about 50 nM to 2000 nM, more preferably about 100 nM to 2000 nM, even more preferably about 100 nM, 200 nM, 500 nM, 1000 nM, 2000 nM, which are used in a continuous round starting from 100 nM in the preferred embodiment.

The introduction of a nucleic acid sequence encoding an antifolate resistant DHFR-variant and preferably one or more polypeptides of the group of SEQ ID NO: 1 to SEQ ID NO: 9 results in a host cell of the human myeloid leukemia origin of the invention and in particular K562, NM-F9 Either DSM ACC2606, NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT- It is surprising that it has been possible to amplify a nucleic acid sequence encoding said protein / antibody molecule or at least a part thereof by incubating with antifolate / methotrexate in a cell or cell line derived from.

Wherein the introduction of a nucleic acid sequence encoding one or more polypeptides of SEQ ID NO: 1 is carried out in a host cell of the present invention and in particular K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- By incubation with methotrexate in a cell or cell line derived from any one of the above-mentioned host cells, such as, but not limited to, NMF9F8, NMF9D8, NMF9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT- It is particularly surprising that amplification of a nucleic acid sequence encoding a portion is possible.

Wherein the introduction of a nucleic acid sequence encoding one or more polypeptides of the group of SEQ ID NOS: 1 to 9 is carried out using the host cells of the invention and in particular K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] In a cell or cell line derived from any one of the above host cells, the host cell is cultured at least twice in a cell or cell line derived from any one of the above-mentioned host cells, such as, but not limited to, -F2, -F9, NM-C-2F5, NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, Adding a nucleic acid sequence encoding said antibody molecule or at least a portion thereof, by increasing the concentration of methotrexate by at least about 50%, more preferably by at least about 100%, in each successive round of incubation with a continuous round of methotrexate It is even more surprising that amplification is possible.

Wherein the introduction of a nucleic acid sequence encoding one or more polypeptides of SEQ ID NO: 1 is carried out in a host cell of the present invention and in particular K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- The host cells are cultured with at least two successive rounds of methotrexate in a cell or cell line derived from any of the above-mentioned host cells, such as NMF9, NMF9, NMF9, NMF9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT- Thereby allowing further amplification of the nucleic acid sequence encoding the antibody molecule or at least a portion thereof by increasing the concentration of methotrexate by at least about 50%, more preferably by at least about 100% in each successive round More amazing.

In a preferred embodiment, the term amplification is stable means that the antibody molecule composition is produced in high yield over at least 35 generations of the host cell division cycle. Amplification of stably introduced nucleic acids or nucleic acids encoding protein / antibody molecules or fractions thereof can be performed as described above and in embodiments using methotrexate.

Additional preferred embodiments are described in the Examples.

In a preferred embodiment of the invention, the host cells of the invention carrying the introduced nucleic acid of the present invention are preferred after at least one round of single cell cloning and selection of cell clones with suitable expression and secretion of the protein / antibody composition Lt; / RTI > Preferably, the single cell cloning and selection of a cell clone with suitable expression and secretion of the protein / antibody composition occurs after at least one round of methotrexate amplification as described herein. In a further preferred embodiment, said cell clone is selected from the group consisting of single cell cloning of methotrexate, preferably an increased concentration of methotrexate, preferably at least about 2-fold concentration of methotrexate, and even more preferably of a further round, Is further amplified by selection of a cell clone with appropriate expression and secretion of the composition. In this preferred embodiment, a cell clone having a particularly high expression yield can be selected.

In accordance with the present invention, the term culturing the host cell under conditions permitting the production of the individual formulation for the antibody molecule composition or protein generally includes one or more nucleic acids encoding a protein / antibody molecule, The host cells of the present invention, including preferred embodiments of the nucleic acid of the present invention, are capable of expressing the protein / antibody molecule in the form of a protein / antibody molecule composition and preferably have the high yields and / / ≪ / RTI > or high homogeneity and are able to be secreted into the medium. The skilled artisan will appreciate that the most suitable conditions and conditions will be selected using suitable media and culture conditions such as, but not limited to, the appropriate time, temperature, pH, gasing, feed, media, media supplement, container or reactor size, Culture conditions can be selected. Those skilled in the art will be able to select and optimize the most suitable conditions. Preferred embodiments are illustrated in the examples, but are not limited thereto.

Culturing the cells of the present invention can be carried out by any conventional culture method for animal cells capable of efficiently producing the desired antibody molecule composition, and can be carried out by any conventional culture method such as batch culture, repeated batch culture, fed-batch culture and perfusion culture. Preferably, fed-batch or batch culture is applied to increase the productivity of the desired polypeptide.

In a further preferred embodiment of the present invention, said culturing is carried out under no-serum conditions and even further preferably under no-protein or no-animal ingredient medium.

The adaptation of the host cells of the invention to the serum-free medium according to the invention is surprisingly rapid and strong. Adaptation can be accomplished, for example, by directly adapting secondary cultured cells in a serum-containing medium to a commercial serum-free medium or by continuous adaptation, wherein direct adaptation to a no-serum medium is preferred, Do. During the adaptation process to the serum-free medium, the viability of the cells is temporarily lowered, which sometimes leads to cell death. Thus, the cells are inoculated with a cell density of 1 x 10 ⁵ to 5 x 10 ⁵ cells / ml, preferably 2 x 10 ⁵ cells / ml, for the adaptation to the serum-free medium, It is desirable to keep it high. After 2 to 7 days of culture, cells with a density of 5 x 10 ⁵ to 10 x 10 ⁵ cells / ml are selected as cells adapted to the no-serum medium. Adherence to serum-free medium can also be achieved by serial dilution of the medium supplemented with FCS with a serum-free medium composition (continuous adaptation). Those skilled in the art will be able to select and optimize the most suitable conditions. Preferred embodiments are described in the Examples, but are not limited thereto.

After the cells of the present invention have been adapted to the serum-free medium, the cloned cell line can be prepared using a limiting dilution method, a colony formation method, etc. in a 96-well plate, Such as, but not limited to, short double-time, faster growth, the possibility of growing under high density, higher yields, higher cloning efficiency, higher transfection efficiency in the case of DNA, The higher expression rate of the composition, the higher activity on the expressed antibody molecule composition, the higher homogeneity of the expressed antibody molecule composition, and / or the greater the magnification. Methods for selecting cells with favorable properties are known to those skilled in the art or disclosed herein.

In accordance with the present invention, the term isolating the corresponding formulations for the antibody molecule composition or protein is generally referred to by the host cell comprising at least one of the nucleic acids encoding the protein / antibody molecule or fractions thereof disclosed herein Means that the expressed protein / antibody molecule composition is obtained using the culture medium after incubation or further incubation or purification of the protein / antibody molecule composition or a portion of the protein / antibody molecule composition by methods known to those skilled in the art. In the context of the present invention, the protein / antibody molecule composition also refers to a portion of the protein / antibody molecule composition that has been hatched for the particular protein / antibody molecule disclosed herein.

In a preferred embodiment, the protein / antibody molecule composition is isolated after incubation, for example, by separating the medium from the cells and / or cell debris by centrifugation techniques.

In a further preferred embodiment of the present invention, the protein / antibody molecule composition of the invention is isolated or further incubated by ultrafiltration, precipitation methods or other concentration methods known to those skilled in the art.

In a further preferred embodiment of the present invention, the protein / antibody molecule composition of the present invention may be used in a method of chromatography, such as, but not limited to, protein A, protein G, anti-antibody isotype antibody, lectin chromatography, The protein / antibody molecule composition is purified by affinity chromatography, which is used according to an affinity substance such as an antigen, or an ion-exchange chromatography known to a person skilled in the art, do.

Additional methods of purifying or hatching a particular sugar form of a protein or protein are known to those skilled in the art and may be used to isolate or further purify, fractionate, or hatch the protein molecular composition or fraction thereof of the invention, Adaptive, optimized, and utilized by those skilled in the art in combination with the methods described herein.

In a preferred embodiment of the present invention, the antibody molecule composition of the present invention, predominantly of IgG, is isolated by protein A chromatography with or without pre-centrifugation.

In another preferred embodiment of the present invention, the antibody molecule composition of the present invention, predominantly IgM, is isolated by anti-IgM antibody chromatography with or without pre-centrifugation.

In another preferred embodiment of the present invention, the antibody molecule compositions of the present invention hatched in the specific sugar form of the antibody molecule are isolated by lectin affinity chromatography with or without pre-ultracentrifugation. Additional methods of purifying or hatching a particular sugar form of a protein or protein are known to those of skill in the art and may be used to isolate or further purify, fractionate, or hatch the antibody molecule composition or fractions thereof of the invention, Adaptive, optimized, and utilized by those skilled in the art in combination with the methods described herein.

In a preferred embodiment, the antibody molecule composition is isolated using a protein A column. In another preferred embodiment, the antibody molecule composition is isolated using an anti-IgM column.

According to the present invention, the term " increased activity " means that the activity of the protein and / or antibody molecule composition of the invention expressed in host cells of human myelogenous leukemia originates from the cell line CHO or CHOdfhr- or CHK or NSO or SP2 / Quot; means higher than the activity of one or more protein / antibody molecule compositions from the same protein / antibody molecule expressed in one or more of the PerC.6 or mouse hybridomas. In a preferred embodiment of the present invention, this means that the activity of the protein and / or antibody molecule composition of the present invention expressed in host cells of human myelogenous leukemia origin is CHOdfhr- [ATCC No. CRL-9096] than the activity of the protein / antibody molecule composition from the same protein / antibody molecule. For antibodies, the increased activity is cytotoxicity or increased binding activity of the increased Fc-mediated cell.

In the sense of the present invention, "activity" is a function or a series of functions performed by a protein molecule in a biological context. In the sense of the present invention, "increased activity ", equivalent terms in the context and grammatical equivalents thereof, are understood as improved or optimal activity with respect to the selected application, wherein the activity is, for example, a minimized or maximized limited value Or may be set to an intermediate value that indicates higher or lower activity as compared to the corresponding protein molecule generated in the prior art. Improved or increased activity in the context of the present invention means an advantageous activity in its biological and / or pharmaceutical sense, which means improved serum half-life, pharmacokinetics, stability, biological activity, binding, antigenicity and / or immunogenicity do. For example, the biological activity of a protein molecular composition could be increased to the extent of reducing adverse biological effects, e. G., To reduced stimulation or reduced immunogenicity of adverse immune effects.

The activity of the protein molecular composition according to the present invention can be determined by a suitable biometric assay capable of measuring the activity of the protein. Those skilled in the art will be able to identify suitable biochemical assays or establish appropriate biochemical assays. In accordance with the present invention, such bioassays include, but are not limited to, cell or molecular or mixed assays including proliferation assays, apoptosis assays, cell adhesion assays, signaling assays, migration assays, cytotoxicity assays, For example, biological in vitro assays. Such biomarkers may be used in animal models or in vivo assays using humans such as biodistribution, pharmacokinetics, pharmacokinetic testing, serum half-life testing, bioavailability testing, efficacy testing, localization studies, Prevention tests are also included. Such bioassays also include stability against chemical, physical, physicochemical, biophysical and biochemical tests such as temperature, shear stress, pressure, pH, conjugation, and the like. Such bioassays also include tests for immunogenicity and / or antigenicity to improve the properties of the protein molecular composition in relation to clinical use. One skilled in the art can determine the activity of the protein molecule composition or the combination of activities described.

In a preferred embodiment, the higher activity of the protein molecule composition may result in higher activity in one or more in vitro models and / or higher activity and / or higher stability in one or more in vivo models as determined by one or more in vivo assays and / / Or longer serum half-life and / or longer bioavailability and / or improved immunogenicity and / or improved antigenicity. Improvements in total activity, herein referred to as higher activity, may result, for example, in lower dosages, longer dosing intervals, less adverse effects, and less toxicity or toxicity of the product on use in humans, or significantly improved drugs depending on the organism And the like.

In a preferred embodiment of the invention, the activity of the protein molecule compositions of the invention expressed in host cells of human myeloid leukemia origin is higher than the activity of one or more protein molecule compositions from the same protein molecules produced in the prior art.

The cytotoxicity of the increased Fc-mediated cells may be enhanced by increased cytotoxicity (ADCC activity) of antibody-dependent cells, complementarity-dependent cytotoxicity (CDC activity), and / or cytotoxicity caused by predation Functional activity). The cytotoxicity of increased Fc-mediated cells, including ADCC activity, CDC activity or predatory activity, can be determined by a variety of methods known to those skilled in the art and some are described in detail in the examples, And the method described in the embodiment is a preferred embodiment of the present invention.

The increased binding activity may be increased by binding to an epitope of an antibody molecule, such as an epitope, an antigen, another polypeptide comprising an epitope, or an epitope of an antibody molecule, or one or more Fc receptor or another effector ligand, Such as Fc-gamma RI, Fc-gamma RII, Fc-gamma RIII, and subgroups therefrom, such as Fc-gamma RIIa, Fc-gamma RIIIa, Fc-gamma RIIIb, or the C1q component of the complementary sequence, Lt; RTI ID = 0.0 > Fc < / RTI > receptor or effector ligand. The increased binding activity may be high affinity, high antigen binding capacity, and / or a greater number of binding sites or combinations thereof. Increased binding activity may result in a variety of effects and activities, including but not limited to the forms of receptor mediated activity disclosed in the background of the present invention. The increased binding affinity, antigen antibody binding capacity and receptor mediated activity can be determined by one or more of a variety of methods known to those skilled in the art and include, but are not limited to, Biacore measurements, Scatchard analysis, ELISA, or RIA Tests to determine the induction of apoptosis in suitable target cells, tests to determine the proliferation of suitable target cells, antagonistic, potent and / or receptor blocking assays of antibody molecule compositions, including but not limited to cell-cell Inhibition of mediated binding, induction of intracellular molecule events. Those skilled in the art will be able to select and / or employ and / or modify combinations of suitable methods or methods for testing binding affinity, antigen-antibody binding, number of binding sites and / or receptor mediated activity.

The increased using the method of the present invention Cytotoxicity caused by Fc - mediated cell cytotoxicity, preferably ADCC activity, CDC activity and / or phagocytosis, and / or an epitope of the antibody molecule or preferably one or more Fc receptors or another effector ligand The ability of an antibody to obtain an increased binding activity for an antibody can generally and / or specifically be tested using the host cells of the invention, and preferred embodiments thereof are disclosed herein.

In a preferred embodiment of the invention, the activity of a protein / antibody molecule composition of the invention expressed in host cells of human myelogenous leukemia origin is determined by expression of the cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / PerC.6 or a mouse hybridoma, preferably CHOdhfr- [ATCC No. CRL-9096]. ≪ / RTI >

In a preferred embodiment of the present invention, the activity of the protein / antibody molecule composition of the invention expressed in host cells of human myelogenous leukemia originates from the cell line CHOdhfr- [ATCC No. More preferably at least 50% higher, more preferably at least 2-fold, more preferably at least 3-fold, even more preferably at least 4-fold higher than the activity of the antibody molecule composition from the same antibody molecule expressed in CRL-9096 More preferably at least 7 times, more preferably at least 10 times, more preferably at least 15 times, more preferably at least 23 times, more preferably at least 30 times, more preferably at least 5 times, more preferably at least 7 times, 50 times or more, more preferably 75 times or more, more preferably 100 times or more, more preferably 150 times or more, more preferably 230 times or more, still more preferably 300 times or more, still more preferably 500 times More preferably at least 750 times, and most preferably more than 1000 times.

Thus, not all biomolecules have to exhibit higher activity, but depending on the use and characteristics of the particular protein molecule composition, some beneficial biological effects may compensate for other less beneficial effects and still be associated with the overall effect of the protein molecule composition Resulting in higher activity. For example, a particular protein molecule composition may result in a much higher activity by binding of its receptor to the cell, thus causing secondary effects such as induction of proliferation, but exhibiting somewhat reduced serum half-life. Higher activity to induce more receptor will then compensate for shorter bioavailability for overall bioactivity. In another embodiment, both shorter half-life and higher activity for receptor induction are both beneficial. In another embodiment, the in vivo activity is not improved, but the in vitro stability improves the production and storage of the protein molecular composition. In another embodiment, a long half-life is required that is not low active.

In a preferred embodiment of the invention, the increased activity of the antibody molecule composition is increased ADCC activity. In another preferred embodiment, the increased activity of the antibody molecule composition is increased CDC. In another preferred embodiment, the increased activity of the antibody molecule composition is increased cytotoxicity caused by phagocytosis. In another preferred embodiment, the increased activity of the antibody molecule composition is an increased binding activity to the epitope. In another preferred embodiment, the increased activity of the antibody molecule composition is an increased binding activity to one or more Fc receptors, preferably Fc [gamma] RIIIA. In a further preferred embodiment, the increased activity of the antibody molecule composition is cytotoxicity of the increased Fc-mediated cell and increased binding activity to the epitope. In a further preferred embodiment, the increased activity of the antibody molecule composition is increased ADCC activity and increased binding activity to the epitope. In a further preferred embodiment, the increased activity of the antibody molecule composition is increased ADCC activity, increased CDC activity, increased binding activity to the epitope, and increased binding activity to one or more Fc receptors. In a further preferred embodiment, the increased activity of the antibody molecule composition is increased ADCC activity, increased cytotoxicity caused by predation, increased binding activity to the epitope and increased binding activity to one or more Fc receptors . In a most preferred embodiment, the increased activity of the antibody molecule composition results in increased ADCC activity, increased cytotoxicity caused by predation, increased CDC activity, increased binding activity to the epitope, and an increase to one or more Fc receptors Lt; / RTI >

According to the present invention, the term " improved homogeneity "means that the protein / antibody molecule composition of the present invention expressed in host cells of human myelogenous leukemia origin of the present invention expresses cell line CHO, or CHOdhfr-, or BHK, Or a greater number of advantageous sugar forms or advantageous forms, as compared to one or more antibody molecule compositions of the same antibody molecule isolated from one or more of SP2 / 0, or PerC.6 or mouse hybridomas, Means that one or more sugar forms (or, preferably, such sugar forms are voluntarily representing at least 1% of the total antibody molecule composition) of less than one of the sugar forms, or antibody molecules. In a preferred embodiment of the present invention, this means that the antibody molecule composition of the present invention expressed in host cells of human myelogenous leukemia origin of the invention expresses the cell line CHOdhfr- [ATCC No. CRL-9096], a smaller number of different sugar forms, or a greater number of advantageous sugar forms or advantageous sugar forms, or fewer one of the antibody molecules, compared to one or more antibody molecule compositions of the same antibody molecule isolated from the same antibody molecule Quot; is meant to include the above form.

One heterogeneity that is particularly problematic for production and human use is sialylation. In a preferred embodiment, the protein / antibody molecule composition of the present invention has improved homogeneity by not containing a sugar form having sialic acid N-glycolyl lyuraminic acid (NeuGc), thus indicating that there is no sugar form in this case Means that the form is less than 1% of all carbohydrate chains that can be obtained from the purified antibody molecule composition and more preferably means that there is no carbohydrate chain that can be detected by methods known to those skilled in the art. Since NeuGc is known to be immunogenic in humans, it is a significant advantage of the host cells of the invention over other production systems such as CHO, NSO, SP2 / 0.

In a preferred embodiment, the protein / antibody molecule composition of the present invention comprises less than 5% sugar form, preferably less than 3%, more preferably less than 1% sugar form and most preferably as described in the Examples Has improved homogeneity with respect to sialylation by virtue of the absence of sugar forms of the protein / antibody molecule composition with sialic acids that can be detected. In a further preferred embodiment, a protein / antibody molecule composition having improved homogeneity with respect to sialylation is produced that is deficient in the precursor pathway of the sugar nucleotide and lacks or reduces CMP-sialic acid, Is obtained using host cells of the human myeloid leukemia origin of the present invention that do not result in sialylation of the carbohydrate sugar chains of the protein / antibody molecule when grown in the medium or result in greatly reduced sialylation. In still further preferred embodiments, the protein / antibody molecule composition having improved homogeneity with respect to sialylation is grown in serum-free medium and incubated with NM-F9 [DSM ACC2606] or NM- D4 [DSM ACC2605] as the host cell of the present invention.

In yet another preferred embodiment, a protein / antibody molecule composition having improved homogeneity with respect to sialylation is contacted with a sugar nucleotide transporter of GMP-sialic acid or a cell with defects in one or more sialyltransferases Is obtained using host cells of the human myeloid leukemia origin of the present invention that do not result in sialylation of the carbohydrate sugar chains of the protein / antibody molecule when grown in a serum-free medium, or result in reduced sialylation. Examples are NM-F9, NM-D4 and GT-2X.

In another preferred embodiment of the present invention, a protein / antibody molecule composition having improved homogeneity with respect to sialylation is obtained using host cells of the human myeloid leukemia origin of the invention having increased degree of sialylation. The degree of sialylation refers to the degree of sialylation when the amount of sialic acid N-acetylneuraminic acid (NeuNc or NeuNAc) on the protein / antibody molecule of the antibody molecule composition is expressed in the cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / 0, or one or more of PerC.6 or mouse hybridomas, preferably CHOdfhr- [ACTT NO. Specific carbohydrate chain or the entire carbohydrate unit at the specific glycosylation site of the protein / antibody molecule, compared to the same amount of protein / antibody molecule in the protein / antibody molecule composition of the same protein / antibody molecule isolated from CRL-9096 Is at least 5%, more preferably at least 15%, more preferably at least 20%, more preferably at least 25%, and even more preferably at least 30%, more preferably at least 5%, more preferably at least 5% %, More preferably at least 35%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80% More preferably at least 90%, more preferably at least 100%, more preferably at least 3 times, even more preferably at least 5 times, more preferably at least 90% 10 times, more preferably at least 25 times, more preferably at least 50 times higher, and most preferably more than 100 times higher. The degree of sialylation can be detected by methods known to those skilled in the art and includes, but is not limited to, immunoblot analysis using lectins such as, for example, SNA, MAL, MAL I or PNA in which the binding is dependent on sialylation of the carbohydrate structure or ELISA , Chemical detection methods such as thiobarbituric acid method, HPLC or mass spectrometry or combinations thereof. Those skilled in the art will be able to select the most suitable method and adapt and optimize it according to the purpose, details of which are disclosed in the examples. Preferred is immunoblot analysis using SNA.

In yet another preferred embodiment, a protein / antibody molecule composition having improved homogeneity with respect to sialylation is administered to host cells of the human myeloid leukemia origin of the invention, preferably K562, NM-F9 [DSM ACC2606] NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X and cells or cell lines derived therefrom, NM-E-2F9 < / RTI > that results in a protein / antibody molecule composition comprising alpha 2-6 linked sialic acid, preferably with increased degree of sialylation, H9D8-E6Q12, GT-2X, and in a more preferred form the protein / antibody molecule composition is obtained using alpha 2-6 and alpha 2 -3 bonded sialic acid.

In another preferred embodiment, a protein / antibody molecule composition having improved homogeneity with respect to sialylation is conjugated to at least one sialyltransferase and alpha 2-6 A host cell of human myelogenous leukemia origin, which may comprise one or more sialyltransferases capable of attaching the binding NeuNAc to the sugar, resulting in a composition of more preferred sugar forms of the protein / antibody molecule in the protein / antibody molecule composition, NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6, NM-D9 [DSM ACC2606], NM-E-2F9, NM- E6Q12, GT-2X and cells or cell lines derived therefrom.

Even in a further preferred embodiment of the invention, the antibody molecule composition with improved homogeneity with respect to sialylation is more stable than the amino acid Asn-297 in the second domain of the Fc region (C-gamma 2 domain) Includes one or more sugar forms of an antibody molecule having one or more carbohydrate chains attached to other glycosylation sites.

In a further preferred embodiment of the former, said antibody molecule composition having improved homogeneity with regard to sialylation is one having an increased degree of sialylation and / or attaching sialic acid to said sugar in an alpha 2-3 linkage A host cell of human myeloid leukemia origin, such as K562, NM-E-2F9, NM-E-2F9, comprising at least one sialyltransferase and at least one sialyltransferase capable of attaching sialic acid to the sugar in an alpha 2-6 linkage H9D8-E6Q12, GT-2X, and cells or cell lines derived therefrom.

In a further preferred embodiment, the former antibody molecule comprises one or more N-glycosylation sites (Asn-X-Ser / Thr, where X can be any amino acid except Pro) and / or one or more O- Region in the sequence of the Fab region.

In a further preferred embodiment, there is provided a method for the production of a sialylated (s) amino acid comprising an antibody molecule comprising at least one carbohydrate chain attached to another glycosylation site of the antibody molecule at the second domain (C gamma 2 domain) Said antibody molecule composition having improved homogeneity with respect to expression of said antibody molecule comprises at least one of the cell line CHO or CHOdhfr-, or BHK, or NS0, or SP2 / 0, or PerC.6 or mouse hybridomas, CHOdfhr- [ACTT No. < / RTI > Has a prolonged serum half-life and / or bioavailability, as measured in one or more mammals, such as mice, rats or preferably humans, rather than the antibody molecule protein of the same antibody molecule isolated from CRL-9096.

The bioavailability of the antibody can be optimized using the present invention. Expression of antibody molecules, particularly in cells with high or very high sialylation (groups 1 and 3 discussed above), may result in antibody compositions with extended bioavailability, but the expression of antibody molecules in cells of group 2 is not equivalent Resulting in an antibody composition with reduced bioavailability. Bioavailability may be tested as known to those skilled in the art, using an animal or preferably a human, and as described in the Examples. The animal includes, but is not limited to, a mouse, a rat, a guinea pig, a dog, or a monkey. Due to human nature, animals with the closest glycosylation to human and the most important sialylation are preferred, and the most preferred is human.

In even more preferred embodiments, the antibody molecule composition having improved homogeneity with regard to sialylation is one or more sialic acid derivatives having an increased degree of sialylation and / or capable of attaching sialic acid to the sugar in an alpha 2-3 linkage Host cells of the human myeloid leukemia origin of the invention, such as K562, NM-F9 [DSM ACC2606 < RTI ID = 0.0 > , NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-F9D8 or NM-H9D8-E6 and cells or cell lines derived therefrom, Or CHOdhfr-, or BHK, or NSO, or SP2 / 0, at least one carbohydrate chain attached to one or more N-glycosylation sites and / or one or more O-glycosylation sites in the sequence of SEQ ID NO: , Or PerC.6 Or a mouse hybridoma, preferably CHOdfhr- [ACTT NO. Has a prolonged serum half-life and / or bioavailability, as measured in one or more mammals, such as mice, rats or preferably humans, rather than the antibody molecule protein of the same antibody molecule isolated from CRL-9096. In a further preferred embodiment, the expressed antibody molecule is erbitux (Cetuximab).

According to the present invention, the term " increased yield " means that the average or maximum yield of a protein / antibody molecule composition of the invention produced in host cells of human myelogenous leukemia originates from the cell line CHO, or CHOdhfr-, or BHK, Or higher than the respective average or maximum yield of one or more protein / antibody molecule compositions from the same protein / antibody molecule upon expression in one or more of the following: SP2 / 0, or PerC.6 or mouse hybridomas. In a preferred embodiment of the present invention, this means that the average or maximum yield of the protein / antibody molecule composition of the present invention produced in host cells of human myelogenous leukemia origin is CHOdfhr- [ACTT NO. CRL-9096] higher than the individual average or maximum yield of one or more protein / antibody molecule compositions from the same protein / antibody molecule upon expression using the murine dhfr gene and methotrexate for amplification in CHOdhfr-. The average and maximum yields are measured in SPR, which reflects the productivity of cells, cell mixtures or cell lines, and can be determined by one skilled in the art and disclosed in the preferred embodiments of the examples.

In a further preferred embodiment of the invention, host cells of human myelogenous leukemia origin, preferably K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM- The average or maximum yield of the protein / antibody molecule composition of the present invention expressed in a cell or cell line derived from NM-F9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT- ACTT No. More preferably at least 15% higher than at least one protein / antibody molecule composition from the same protein / antibody molecule expressed using the murine dhfr gene and methotrexate for amplification in CHOdhfr- , More preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35%, more preferably at least 45%, even more preferably at least 50% , Preferably at least 55%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100% Is at least 3 times, more preferably at least 4 times higher, and most preferably more than 5 times higher.

The invention provides a kit comprising (a) a sequence encoding a protein, preferably an antibody molecule or at least a portion thereof, as disclosed herein, and (b) one or more sequences encoding a sequence from the group of SEQ ID NOS: Nucleic acids are additionally provided .

In a preferred embodiment of the invention, the nucleic acid of the present invention comprises (a) a sequence encoding a protein, preferably an antibody molecule or at least a portion thereof, as disclosed herein, and (b) a sequence encoding SEQ ID NO: 1.

In a further preferred embodiment of the present invention, the disclosed nucleic acids of the present invention further comprise a sequence encoding a selectable marker, preferably a polypeptide encoding a polypeptide inducing antibiotic resistance of the host cell into which the nucleic acid is introduced, But are not limited to, neomycin or promicin.

In further preferred embodiments of the present invention, the disclosed nucleic acids of the invention further comprise one or more sequences of one or more of the gene elements disclosed herein.

The present invention encompasses any human myelogenous or myeloid precursor cell or cell line obtainable from a human myelogenous leukemia origin or a leukemia patient, or any myelogenous or myeloid precursor cell or cell line obtainable from a human donor, or one of human myelogenous leukemia origin Or any human myeloid or myeloid precursor cell or cell line obtainable from a human myelogenous leukemia origin or a leukemia patient or any myelogenous or myeloid precursor cells obtainable from a human donor Or host cell of a cell, cell or cell line obtained by fusing a cell line with another cell of human or animal origin, including but not limited to a protein / antibody molecule introduced into the cell It is a B-cell, CHO cell containing one or more nucleic acids encoding a portion thereof.

The present invention provides a host cell of the disclosed invention comprising at least one nucleic acid encoding a protein / antibody molecule or at least a portion thereof.

In a preferred embodiment, the present invention is directed to a host cell selected from the group consisting of host cells K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- E- 2F9, NM- NM-H9D8-E6Q12, GT-2X, or cells or cell lines derived from any of the above host cells, which are preferably grown under serum-free conditions, from which the protein / More preferably wherein the nucleic acid encoding the antibody molecule is introduced under serum-free conditions, which comprises at least one nucleic acid encoding a protein / antibody molecule or a portion thereof, preferably a protein / antibody A nucleic acid comprising a sequence encoding a molecule or at least a portion thereof.

The present invention provides a host cell of the presently disclosed invention comprising at least one nucleic acid encoding a polypeptide of SEQ ID NO: 1 to SEQ ID NO: 9, preferably at least one polypeptide of SEQ ID NO:

The present invention relates to a host of the presently disclosed invention comprising at least one nucleic acid encoding an antibody molecule or a portion thereof and at least one nucleic acid encoding a group of SEQ ID NO: 1 to SEQ ID NO: 9, preferably at least one polypeptide of SEQ ID NO: Lt; / RTI >

In a preferred embodiment, the present invention is directed to a host cell selected from the group consisting of host cells K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- E- 2F9, NM- NM-H9D8-E6Q12, GT-2X, or cells or cell lines derived from any of the above host cells, which are preferably grown under serum-free conditions, from which the protein / Nucleic acid encoding the protein / antibody molecule is introduced under serum-free conditions, which is separated by one or more nucleic acids encoding the protein / antibody molecule or a portion thereof, preferably the protein disclosed herein / Antibody molecule or at least a portion thereof, and one or more sequences encoding a sequence from SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: 1.

In a further preferred embodiment, the invention provides a host cell as described above, wherein the encoded antibody molecule is an antibody of WO2004 / 065423.

In a further preferred embodiment, the invention provides a host cell as described above, wherein the encoded antibody molecule is an antibody selected from the group consisting of the antibody PancoMab [ Cancer Immunol Immunother . 2006 Nov; 55 (11): 1337-47. Epub 2006 Feb. PankoMab: a potent new generation anti-tumor MUC1 antibody. Danielczyk et < RTI ID = 0.0 > al., ≪ / RTI > preferably a chimeric form of fancomaps having all human constant domains, and more preferably humanized fancomaps.

The present invention further provides a protein / antibody molecule composition having increased activity and / or increased yield and / or improved homogeneity and complete human glycosylation produced by any of the methods of the invention disclosed herein.

In a preferred embodiment, the invention provides a protein / antibody molecule composition produced by any of the methods of the invention, which expresses the cell line CHO, or CHOdhfr-, or BHK, or NSO, or SP2 / 0.0 > CHOdhfr- < / RTI > [ATCC No. Or improved homogeneity and / or improved homogeneity produced by any of the methods of the present invention, as compared to the protein / antibody molecule composition of the same protein / antibody molecule isolated from a human Glycosylation.

The expressed protein molecule or portion thereof may be any protein or protein portion or protein fragment. The expressed antibody molecule or portion thereof may be any antibody or antibody portion or antibody fragment.

The protein molecular composition of the present invention can be used for the prophylactic and / or therapeutic treatment of diseases such as leukemia, neutropenia, hemopneumonia, cancer, bone marrow transplantation, hematopoietic diseases, infertility and autoimmune diseases. Therapeutic applications of protein molecular compositions known to those skilled in the art are very broad. For example, G-CSF is an important treatment for the treatment of neutropenia, a reduction in life-threatening neutrophils as a result of chemotherapy in patients with leukemia. GM-CSF is especially used for the treatment of relatively elderly AML patients for rapid recovery from neutropenic leukopenia after chemotherapy. GM-CSF was further approved as a therapeutic agent for various applications in bone marrow transplantation and as a therapeutic agent for the mobilization of peripheral blood stem cells. In addition, there are several clinical applications of GM-CSF currently under study, such as for treating HIV and cancer. Certain diseases of the hematopoietic system are treated with EPO, and IFN-beta is currently an important therapeutic agent for the treatment of multiple sclerosis, an autoimmune disease. Another example is FSH, which is widely used to treat male and female infertility. hCG is also applied to treat infertility, but focuses on women's anovulation. hGF has clinically proven benefits such as decreased body fat and increased muscle tissue.

The protein molecular composition of the present invention is useful for the prophylactic and / or therapeutic treatment of diseases selected from the group including leukemia, neutropenia, hematopoietic, cancer, bone marrow transplantation, hematopoietic diseases, infertility and autoimmune diseases And may be used for the production of pharmaceuticals.

In a preferred embodiment of the present invention the antibody molecule is selected from the group consisting of psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, autoimmune disease, SLE, multiple sclerosis, autoimmune blood disease, asthma, allergy, graft versus host disease, It is an antibody molecule that recognizes viruses such as surgery, myocardial infarction, RSV, HIV, Hep B or CMV, cancer, sarcoma, CLL, AML or NHL.

Even in a preferred embodiment of the invention, the antibody molecule being expressed is an antibody molecule which recognizes cancer, tumor or metastasis, at least one cancer cell or tumor cell in at least one person, preferably an ear-nose- Neoplasms of the central nervous system, cancerous diseases or tumor diseases during infancy, lymphomas, leukemias, adrenal organisms, lymphomas, lymphomas, adrenocortical organisms, (CUP syndrome), peritoneal carcinomatosis, immunosuppression-associated malignancy, and / or tumor metastasis.

In a preferred embodiment of the present invention, the expressed antibody or a portion thereof is an anti-MUC1 antibody.

In a further preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the invention is an antibody rituximab, herceptin, erbuxox, campath 1H or an antibody derived therefrom.

In an even more preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/050707, more preferably an antibody described in WO 2004/065423, Desirably, it is desirable to use a monoclonal anti-tumor antibody (see Cancer Immunol Immunother. 2006 Nov; 55 (11): 1337-47. Epub 2006 Feb. PankoMab: a potent new generation anti-tumor MUC1 antibody, Danielczyk et al) Quot; is a chimeric version thereof, including all human constant domains, and even more preferably is a humanized antibody thereof.

In an even more preferred embodiment of the invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention comprises any complete antibody of the invention, preferably rituximab, herceptin, erbuxox, more preferably Preferably the K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- An antibody molecule isolated from a cell or cell line derived from any one of the above host cells, such as, but not limited to, E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT- The composition does not contain any detectable NeuGc. In general, the same explanation applies to proteins.

In an even more preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention may comprise any complete antibody molecule, or an antibody molecule comprising the C-gamma 2 domain of the invention, , Preferably a K562, NM-F9 [DSM ACC2606], or an immunogenic fragment thereof, which is an antibody described in WO 2004/065423, most preferably a fancomap, NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, or any of the above host cells An antibody molecule composition isolated from a derived cell or cell line comprises at least one sugar form with alpha 2-6 sialic acid. In general, the same explanation applies to proteins.

In an even more preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention may comprise any complete antibody molecule, or an antibody molecule comprising the C-gamma 2 domain of the invention, 2-F9, NM-C-2F5, NM-C-2F5, NM-C-2F5 and NM-C-2F5, and the antibodies described in WO 2004/065423, most preferably, The antibody molecule composition isolated from a cell or cell line derived from any of the NM-H9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, or any of the host cells includes alpha 2-6 sialic acid And the sialic acid is detectable by immunoblot assay using the lectin SNA described in the Examples. In general, the same explanation applies to proteins.

In another preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention may be any antibody molecule, preferably rituximab, herceptin, erbitabus, campat 1H, An antibody as described in WO 2004/065423, most preferably a fancomap, and an antibody isolated from the host cell NM-F9 or NM-D4 of the present invention following incubation in a serum-free medium and more preferably in a protein- The molecular composition exhibits improved homology without any detectable sialic acid. In general, the same explanation applies to proteins.

In a further preferred embodiment of the invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody rituximab, Herceptin, Kampatsu 1H, or an antibody derived therefrom, The isolated antibody molecule composition was purified from the cell line CHOdhfr- [ATCC No. CRL-9096] that is at least 4 times higher than the activity of the antibody molecule composition from the same antibody molecule.

In an even more preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomap, wherein the antibody isolated from the host cell of the invention The molecular composition contains the cell line CHOdhfr- [ATCC No. NM-E-2F9, NM-C-2F5, NM-C-2F5, NM-C-2F5, At least 4-fold increased ADCC activity when produced in at least one of the cells or cell lines derived from any one of the above-mentioned host cells, such as -H9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT- .

 In another preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomap, and the host cells K562, NM-F9 NM-H9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, or any of the above-described host cells (DSM ACC2606, DSM ACC2605, NM-E-2F9, NM- An antibody molecule composition derived from a cell or cell line derived from any one of the cell lines can be obtained from the cell line CHOdhfr- [ATCC No. Has an increased binding activity for its epitope at least 50% higher, preferably 2-fold higher, than the activity of the antibody molecule composition from the same antibody molecule expressed in CRL-9096.

In another preferred embodiment of the invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomaps, serum-free medium, and more preferably, The antibody molecule compositions isolated from the host cells NM-F9 or NM-D4 after incubation in a protein-free medium have improved homology without any detectable sialic acid. In general, the same explanation applies to proteins.

In another preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomap, and is a host cell K562, NM-F9 [DSM ACC2606 , NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8 or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, or any of the above host cells The antibody molecule composition isolated from a cell or a cell line derived from the cell line CHOdhfr- [ATCC No. 1]. Having at least 10% more sialic acid than the antibody molecule composition from the same antibody molecule expressed in CRL-9096. In general, the same explanation applies to proteins.

In another preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomaps, and the cell line CHOdhfr- [ATCC No. Has a higher degree of undetectable sialic acid and has improved homology when expressed in NM-F9 or NM-D4 as compared to the antibody molecule composition from the same antibody molecule expressed in CRL-9096. In general, the same explanation applies to proteins.

In another preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomap, wherein the antibody molecule isolated from the host cell of the invention The composition has improved homology as described above, and the cell line CHOdhfr- [ATCC No. CRL-9096] that is at least 4 times higher than the activity of the antibody molecule composition from the same antibody molecule expressed in the cell.

In the most preferred embodiment of the present invention, the antibody molecule encoded by the nucleic acid or nucleic acids of the present invention is an antibody described in WO 2004/065423, more preferably a fancomaps.

The present invention provides a host cell for producing a protein / antibody molecule composition having increased activity and / or increased yield and / or improved homology and sufficient human glycosylation in terms of the present invention as elsewhere herein , Any of the human bone marrow or bone marrow precursor cells or cell lines available from a cell, cell or cell line of human myeloid leukemia origin, or from a patient with leukemia, or any bone marrow or bone marrow precursor A cell or cell line, or a mixture of cells or cell lines comprising at least one cell of human myelogenous leukemia origin, or a cell or cell line derived therefrom as described elsewhere herein, or at least one of the above-mentioned cells A host cell, which is a mixture of cells or cell lines, to provide.

The present invention also provides a method for producing a protein / antibody molecule composition having increased activity and / or increased yield and / or improved homology, and sufficient human glycosylation in terms of the present invention as described elsewhere herein, Wherein the host cell is a cell, cell or cell line of human myeloid leukemia origin, or any bone marrow or bone marrow precursor cell or cell line available from a patient with leukemia, or any bone marrow available from a human donor, or The present invention provides a host cell, which is any cell, cell or cell line obtained by fusing a bone marrow precursor cell or cell line to another cell of human or animal origin, but not limited to a B cell or a CHO cell.

In a preferred embodiment, the cell or cell line KG1, MUTZ-3, K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- The host cell of the present invention, which is a cell or cell line derived from H9D8-E6, NM-H9D8-E6Q12, GT-2X, or a cell or cell line derived therefrom, or a cell or a mixture of cell lines comprising at least one of the above- Are provided to produce protein / antibody molecule compositions having increased activity and / or increased yield and / or improved homology in the context of the invention described elsewhere herein.

In a further preferred embodiment, the cell or cell line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM- The host cell of the invention, which is a cell or cell line derived from NM-H9D8-E6Q12, GT-2X, or a cell or cell line derived therefrom or those described elsewhere herein, Lt; RTI ID = 0.0 > and / or < / RTI > improved homology.

In a further preferred embodiment, the cell or cell line NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8 or NM- The host cells of the invention, which are H9D8-E6Q12, GT-2X, or cells or cell lines derived therefrom, have increased activity and / or increased yield and / or improved homology in the context of the invention described elsewhere herein ≪ / RTI > protein / antibody molecule composition.

In yet a further preferred embodiment, one or more compounds selected from the group consisting of KG1, MUTZ-3, K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM- A cell or cell line derived from H9D8-E6, NM-H9D8-E6Q12, GT-2X, or a cell or cell line derived therefrom which has been grown under serum-free conditions, and preferably a protein / The host cell of the present invention wherein the encoding nucleic acid is introduced into these cells and the antibody molecule composition is such that it is isolated under serum free conditions may be used in an amount sufficient to achieve increased activity and / / Or < / RTI > protein / antibody molecule composition having improved homology.

In yet a further preferred embodiment, K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM- The host cell of the present invention, which is a cell or cell line derived from H9D8-E6Q12, GT-2X, or a cell or cell line derived from those elsewhere herein grown under serum-free conditions, / RTI > is provided to produce a protein / antibody molecule composition having increased activity and / or increased yield and / or improved homology in terms of < / RTI >

In yet a further preferred embodiment, K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM- A cell or cell line derived from H9D8-E6Q12, GT-2X, or a nucleic acid encoding a protein / antibody molecule is introduced into these cells and the protein / antibody molecule composition is isolated under serum- The host cell of the invention, which is a cell or cell line derived from those elsewhere herein, is a protein / antibody having increased activity and / or increased yield and / or improved homology in the context of the invention described elsewhere herein To provide a molecular composition.

In the most preferred embodiment, the cell or cell line NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C- 2F5, NM- H9D8 or NM- A cell or cell line derived from those elsewhere herein grown under E6Q12, GT-2X, or serum-free conditions, and preferably a nucleic acid encoding a protein / antibody molecule are introduced into these cells and the protein antibody molecule composition The host cells of the present invention, which are those isolated under this serum-free condition, can be used in combination with a protein / antibody molecule composition having increased activity and / or increased yield and / or improved homology in terms of the invention elsewhere herein / RTI >

The present invention further provides protein / protein compositions separated by any of the methods of the invention described elsewhere herein.

The present invention provides any of the methods of the invention described elsewhere herein having increased activity and / or increased yield and / or improved homology and sufficient human glycosylation as described herein and in the context of the present invention Lt; RTI ID = 0.0 > molecular < / RTI > composition.

In a further preferred embodiment of the invention, the protein molecule or portion thereof has a size of at least 10 kDa, preferably at least 15 kDa, more preferably at least 20 kDa, more preferably at least 25 kDa More preferably at least 30 kDa, more preferably at least 35 kDa, more preferably at least 40 kDa, even more preferably at least 45 kDa, even more preferably at least 50 kDa More preferably at least 55 kDa, more preferably at least 60 kDa, more preferably at least 65 kDa, even more preferably at least 70 kDa, even more preferably at least 75 kDa , More preferably at least 80 kDa, more preferably at least 85 kDa, even more preferably at least 90 kDa, Is at least 95 kDa in size, more preferably at least 100 kDa in size, more preferably at least 105 kDa in size, more preferably at least 110 kDa in size, more preferably at least 115 kDa in size, Preferably has a size of at least 120 kDa, more preferably at least 125 kDa, more preferably at least 130 kDa, more preferably at least 135 kDa, even more preferably at least 140 kDa, Is at least 145 kDa in size, more preferably at least 150 kDa in size, more preferably at least 155 kDa in size, more preferably at least 160 kDa in size, more preferably at least 165 kDa in size, Is at least 170 kDa in size, more preferably at least 175 kDa in size, more preferably at least 180 kDa in size, Also is the size of 185 kDa, and more preferably has a size of at least 190 kDa in size, more preferably of at least 195 kDa in size, and most preferably at least 200 kDa.

In a preferred embodiment of the invention, the protein molecular composition is derived from any of the protein molecules of the cytokine and group of receptors thereof, such as the tumor necrosis factors TNF-alpha and TNF-beta; Renin; Human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipid protein; Alpha-1-antitrypsin; Insulin A-chain and B-chain; Gonadotropin, such as FSH, luteinizing hormone (LH), thyrotropin, and human chorionic gonadotropin (hCG); Calcitonin; Glucagon; Coagulation factors such as Factor VIIIC, Factor IX, Factor VII, tissue factor and vimentin gene; Anti-coagulation factors such as protein C; Atrial Sodium Excretion Promoter; Pulmonary surfactants; Plasminogen activators such as eukaryotic, plasminogen activators of the human urine and tissue type; Bombesh Shin; Thrombin; Hematopoietic growth factors; Enkephalinae; Human macrophage inflammatory protein; Serum albumin, such as human serum albumin; Mullerian-inhibiting substances; Lt; / RTI > A-chain and B-chain; Prolaxin; Mouse hypertonic tropin-related peptides; Vascular endothelial growth factor; Hormone or growth factor receptor; Integrin; Proteins A and D; Rheumatic factor; Neurotrophic factors such as bone-derived neurotrophic factor, neurotrophin-3, -4, -5, -6 and nerve growth factor-beta; Platelet derived growth factor; Fibroblast growth factor; Epidermal growth factor; Growth factors that are switched, such as TGF-alpha and TGF-beta; Insulin-like growth factor-I and -II; Insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 and CD-19; Erythropoietin (EPO); Bone inducer; Immunotoxin; Osteogenic protein; Interferons such as interferon-alpha, -beta, and gamma; Colony stimulating factors (CSF's) such as M-CSF, GM-CSF and G-CSF; Interleukins (IL's), such as IL-1 through IL-12; Superoxide dimutase; T-cell receptors; Surface membrane protein; Decay accelerating factor; Antibodies and immunoconjugates; Glycophorin A; MUC1. ≪ / RTI >

In a more preferred embodiment of the present invention, the protein molecular composition comprises protein molecules of the group consisting of glycophorin A, EPO, G-CSF, GM-CSF, FSH, hCG, LH, interferon, ≪ / RTI >

A glycol protein or protein composition obtainable by the production process according to the invention is also provided. The protein preferably has the glycosylation properties as defined in claim 27.

Preferably, the protein composition is an antibody molecule composition. The present invention also provides isolated antibody molecules that are isolated by any of the methods of the invention described herein having increased activity and / or increased yield and / or improved homology, both in the context of the present invention and elsewhere herein The composition is further provided.

Examples of such antibodies include antibodies to Ganglioside GD3, human interleukin-5 receptor alpha-chain, HER2, CC chemokine receptor 4, CD20, CD22, neuroblastoma, MUC1, epidermal growth factor receptor (EGFR).

In a preferred embodiment of the present invention, the antibody molecule composition is selected from the group consisting of mulloomap, daclizumab, basilic immap, abciximab, rituximab, herceptin, gemtuzumab, alemtuzumab, Cetuxi Map (Ervitux), Bebeshima Map, Tositomo Map, Pablo Zypom, InfixiMap, Ichulli Zypom, Efratu Zypom, O'Malley Zypom, Ephalli Zypom, Adalmum Map, Kampatsu -1H, C2B8, Pano Derived from any of the antibody molecules of the group consisting of Rex, Blevarex, Simullect, Antova, OKT3, Genapax, Leopro, Sinagis, Ostivir, Protovir, Obarex,

In a more preferred embodiment of the present invention, the antibody molecule composition comprises at least one of rituximab, herceptin, anti-CCK moiety receptor 4 antibody KM2160, campsat-1H, C2B8, erbuxox, anti- ≪ / RTI > group of antibody molecules. In an even more preferred embodiment of the present invention said antibody molecule composition comprises an antibody molecule of the group described in WO 2004/065423, more preferably a fancomap, more preferably a chimeric form thereof, and even more preferably its humanized Lt; / RTI > antibody molecule.

Also provided is a protein or protein composition comprising an amino acid sequence DTR, wherein the protein is an antibody that binds to a MUC1 epitope and which can be obtained by the production method of the present invention.

MUC-1 is an established tumor marker that is expressed on various epithelial tumors and is a potential tumor target. MUC-1 is a highly O-glycosylated large trans membrane glycoprotein. The extracellular portion consists of a variable number of 20 to 120 tandem repeats (TR), each composed of 20 amino acids with five potential O-glycosylation sites. MUC 1 is not only expressed in epithelial tissues but also in hematopoietic cells. A number of antibodies are known which bind to the DRT motif of MUC 1, which are suitable proteins / antibodies in the context of the present invention (see Karsten et al., 1998). Karsten describes a novel carbohydrate derived morphological epitope on MUC 1 (TA MUC) of the ... PDT ^* RP ... structure, where T ^* is O-glycosylated. Glycans present at this site are themselves tumor-specific carbohydrate structures.

Thus, it may be desirable to use an MUC antibody that is capable of discriminating between a TA MUC tumor epitope and a non-glycosylated epitope. A suitable antibody is a fancomab antibody so long as it can specifically recognize the glycosylated TA MUC epitope. Its production has been described in detail in Danielczyk et al 2006, which is incorporated herein by reference in its entirety (Pancomaps: a novel, potent anti-tumor MUC-1 antibody). Antibody codomaps or variants thereof are competitively bound to the same TA-MUC 1 epitope because the parental Cognom antibody is preferably used. Such antibody variants have one or more of the following characteristics:

- bound to an epitope comprising at least the amino acid sequence PDTRP;

0.0 > Gal-NAc < / RTI > alpha in the PDTRP sequence, but when the same peptide is not glycosylated, it binds to a short MUC peptide of 30 amino acids comprising 1,5 TR;

An additional length effect of more than 25, preferably of 28 (most preferably a ratio of 29.5);

- exhibit low binding or even no binding to cells of the hematopoietic system (see Danielczyk et al 2006, which is incorporated herein by reference in the context of detection methods);

- has a high affinity for tumor cells at least about _Kass = 0.2 - 1 x 10 ⁹ M ^-1 as measured by Scatchard plot analysis.

Suitable examples of individual variants are provided in the examples. Antibodies can be murine, origin, chimeric or humanized.

Antibodies conjugated to the TA-MUC 1 epitope, preferably the Panchoplad antibodies or antibodies panco 1 and panco 2 disclosed herein, have one or more of the following glycosylation characteristics:

(i) CHOdhfr- < / RTI > CRL-9096], the amount of carbohydrate in a particular glycosylation site of the antibody molecule in the entire carbohydrate structure or in the antibody molecules of the antibody molecule composition, as compared to the same amount of antibody molecule in one or more antibody molecule compositions of the same antibody molecule, Structure, an increased degree of sialylation with an amount of N-acetylneuraminic acid of at least 15% higher;

(ii) CHOdhfr- < / RTI > CRL-9096], the amount of carbohydrate in a particular glycosylation site of the antibody molecule in the entire carbohydrate structure or in the antibody molecules of the antibody molecule composition, as compared to the same amount of antibody molecule in one or more antibody molecule compositions of the same antibody molecule, Structure, a higher degree of galactosylation with a G2 structure in an amount of at least 5% higher;

(iii) contains a detectable amount of bisecting GlcNAc;

(iv) does not have a hybrid or high mannose structure or is less than 2%.

The individual glycosylation patterns result in the following surprising and favorable activity patterns:

(i) CDC activity greater than 15% greater than the activity of the same antibody expressed in CHO cells;

(ii) an extended serum half-life of 2-fold (greater than 1.5-fold) compared to antibodies that do not have detectable sialylation;

(iii) Cell line CHOdhfr- [ATCC No. 1]. CRL-9096] cytotoxicity of increased Fc-mediated cell at least 2-fold higher than the cytotoxicity of Fc-mediated cells of one or more antibody molecule compositions from the same antibody molecule.

Individual antibodies can be obtained by production in a cell line according to the invention which provides high sialylation and degree of galactosylation, but preferably low fucosylation. Suitable examples are NM-H9D8 and NM-H9D8-E6.

The following tables and figures illustrate the present invention.

The following Tables 1 and 8 and Figures 1 to 17 illustrate the present invention.

Table 1: Yield of chimeric fancomaps expressed in CHOdhfr- and NM-F9 cultured in medium supplemented with FCS.

Table 2: Yield of chimeric pancomapped expressed in NM-H9D8 [DSM ACC2806] cultured in serum-free medium.

Table 3: Quantification of sialic acid content in vancomaps and cetuximaps: The antibody is quantified by integrating the peak area produced by the indicated cell line and the reverse peak of the DMB labeled sialic acid variant obtained by reverse phase chromatography. NeuGc and NeuAc are differentiated using sialic acid standards.

Table 4: Quantification of different charged structures in Panco 1 and Fancomaps: The 2-AB labeled N-glycans were treated with anion exchange chromatography (Asahi-PAK column) and the peaks corresponding to the different charge structures were quantitated by integration Respectively.

Table 5: The degree of galactosylation of antibody panco 1 and fancomaps was determined by amino-phase HPLC (Luna-NH2-column) DP of 2-AB labeled glycans. The peak was quantified by integration and the underlying glycan structure was analyzed by mass spectrometry.

Table 6: Quantification of triantennary and biantennary + bisector structures in Panco 1 and Pancomap. Potential bisecting GlcNAc containing fractions from amino-HPLC was collected and treated with reverse phase chromatography (RP18-column). Thereby, the triantennary and the biantennary + bisector structure can be distinguished. The degree of fucosylation of the antibody was determined by amino-phase HPLC (Luna-NH2-column) of glycan with 2-AB labeling. The peak was quantified by integration and the underlying glycan structure was analyzed by mass spectrometry. The fucosylated and fucosylated structure was determined and the integrated peak area was determined.

Table 7: Yield of hFSH expressed in CHOdhfr- and GT-2x cultured in medium supplemented with FCS (CHOdhfr-) or no-serum medium.

Table 8: Yield of hFSH expressed in NM-H9D8 [DSM ACC5 number] cultured in serum-free medium.

Table 9: Suitable glycosylation and combinations of activities obtainable by the process according to the invention.

Table 10: Values obtained for bisecting GlcNAc and fucose in different cell lines.

Figure 1: expression of fut8 mRNA of NM-F9, NM-D4, and NM-H9D8 [DSM ACC2806] cells. HepG2 cells were used as a control.

Figure 2: Europium emission assay using cetuximap isolated from NM-H9D8-E6 cells, CHOdhfr- cells or SP2 / 0 cells against LS174T cells as target cells.

The assays were incubated with antibodies at a concentration of 0-100 ng / ml with an effector to target cell ratio of 50: 1.

Figure 3: The ADCC activity of chimeric fancomaps isolated from NM-F9 is ~ 5-fold higher than the chimeric fancomaps isolated from CHOdhfr- cells.

Figure 4: Eurufumer emission assay using chimera panco 1 isolated from NM-H9D8-E6 cells, CHOdhfr- cells or NM-H9D8 cells against ZR-75-1 cells as target cells.

Assays were incubated for 4 hours with antibodies at a concentration of 0 to 1 [mu] g / ml with an effector to target cell ratio of 80: 1.

Figure 5: effector versus target cell ratio of 50: 1 and o.n. ADCC activity of chimeric panco 2 in europium release assay for ZR-75-1 cells after incubation. Samples were incubated in triplicate.

Figure 6: effector-to-target cell ratio of 50: 1 and 10.000 target cells per well. ADCC activity of chimeric panco 2 in europium release assay for ZR-75-1 cells after incubation. Samples were incubated in triplicate.

Fig. 7: CDC test using ZR-75-1 with chimeric facial map.

Figure 8: The binding activity of chimeric fancomaps isolated with synthetic glycosylated MUC1 30-mer peptides from NM-F9 is about 50% higher than the binding activity of chimeric fancomaps isolated from CHOdhfr- cells.

Figure 9: The binding activity of chimeric panco 2 isolated from synthetic-glycosylated MUC1 30-mer peptides from GT-2x and NM-H9D8 is approximately 50% higher than the binding activity of chimeric panco 2 isolated from CHOdhfr- cells.

Figure 10: Western blot analysis is performed to identify differently sialylated heavy chains of antibody molecule compositions expressed in CHOdhfr-, NM-F9, or NM-H9D8 [DSM ACC2806]. The protein is visualized by transfection with nitrocellulose and secondary anti-human IgG antibody (Figure 10A) or SNA (Figure 10B) detecting 2-6 sialylation.

Figure 11: Western blot analysis is performed to identify differently sialylated heavy chains of antibody molecule compositions expressed in CHOdhfr- or NM-H9D8. The protein is visualized by SNA transfected with nitrocellulose and detecting 2-6 sialylation.

Figure 12: ELISA analysis is performed to identify differently sialylated antibody molecule compositions expressed in CHOdhfr-, GT-2x, NM-H9D8 or NM-H9D8-E6.

Figure 13: ELISA analysis is performed to identify differentially sialylated antibody molecule compositions of cetuxime mapped on CHOdhfr-, NM-F9, or NM-H9D8. Sialylation is analyzed by (A) SNA which detects alpha 2-6 sialylation with or without neuraminidase treatment and (B) MAL I which detects alpha 2-3 sialylation.

Figure 14: Chimera antibodies isolated from CHOdhfr-, NM-F9, NM-H9D8 or NM-H9D8-E6 cells Dot blot stained with SNA of Panco 1 and Panco 2.

Figure 15: Chimeric fancomaps isolated from NM-H9D8 cells are longer available in nude mouse serum than those isolated in NM-F9.

Figure 16: SDS-PAGE analysis of hFSH generated from NM-H9D8 (lane 1) and GT-2x (lane 2). 5 [mu] g of purified hFSH was separated by SDS-PAGE per lane under reducing conditions and stained with Coma Brilliant Blue. Markers range from 21 to 108 kD.

Figure 17: Western blot analysis is performed to identify differently sialylated hFSH molecular compositions. 1 mu g of hFSH molecules from CHO (lane 1), NM-H9D8 (lane 2) and GT-2x (lane 3) were separated by SDS-PAGE under reducing conditions in 10% acrylamide gel. Protein was transfected with nitrocellulose and SNA was visualized to detect 2-6 sialylation.

Figure 18: Represents an IgG antibody, wherein the N-glycan is covalently attached to the Asn 297 residue conserved in the C _H 2 domain of Fc. As indicated, additional N-linked oligosaccharides may be present in the Fab domain, which may affect the binding activity of the antibody. The glycan structure is shown in only half of the antibody. Fc carbohydrates are mainly a branched chain structure within two C _H 2 domains and one arm / antenna of each oligosaccharide interacts with the hydrophobic region of the C _H 2 domain. Structural analysis of polyclonal and myeloma human IgG has demonstrated that Fc contains various amounts of base biantennary core structures as evidenced in FIG. The meanings of the symbols are given in the corresponding tables. The core structure may contain 0 (G0), 1 (G1) or 2 (G2) terminal galactose residues and / or bisecting GlcNAc and / or fucose residues in the adjacent GlcNAc. Additional fucose molecules may also be present in other GlcNAc residues or galactose residues. As sialic acid has been reported to adversely affect ADCC, diversity is also increased as terminal sialic acid is present or absent depending on the nature of the antibody.

Example  One

In a cell fut8  Analysis of Expression

RNA was analyzed by standard procedures (RNeasy-Mini-Kit, Qiagen), NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-H9, K562, NM-H9D8 [DSM ACC2806] ACC] and HepG2 (control) cells. mRNA was isolated using magnetic beads technology (HDynabeads® Oligo (dT) 25H, Invitrogen) according to the manufacturer's instructions. For first strand P ^P cDNA synthesis, 50 μl of each sample of bead suspension and Omniscript Reverse Transcriptase (Qiagen) were used according to the manufacturer's instructions. For subsequent P ^P RT-PCR reactions, a 212 bp fragment was generated using 5 μl of the cDNA product P ^P and a specific fut 8 primer. A 240 bp fragment was generated using an actin specific primer as a control. The resulting PCR-product (s) P ^P was analyzed on a 1.5% gel.

NM-F9, NM-D4, NM-H9, K562, NM-H9D8 and GT-2x express mRNA for fut 8.

Figure 1 shows, by way of example, fut8 mRNA expression of NM-F9, NM-D4, and NM-H9D8.

Example  2

K562  Saccharification of cells ( glycoengineering )

The glycosylation of K562 cells and the production of NM-D4 and NM-F9 cell lines are disclosed in EP1654353.

NM-H9 cells with high sialylation potential, and cells or cell lines derived therefrom, were generated as follows. K562 cells were treated with an alkylating agent, ethyl methanesulfonate, to effect random mutagenesis. K562 cells per sample were washed in PBS and seeded with 10P ^6P cells overnight at 37 ° C and 5% COB _2B for 1 ml of cell culture medium supplemented with EMS (0.1 mg / ml, ethyl methanesulfonate, Sigma-Aldrich) . Cells were washed and fresh medium was provided. Cell viability was measured every 2 days by trypan blue staining and the cells were analyzed by immunocytochemical staining.

Subsequently, cells showing a novel phenotype of high TF expression were selected by TF-specific antibody means. K562 cells were washed with B-PBS (0.5% BSA in PBS) and incubated with 50 [mu] l of supernatant of monoclonal antibody A78-G / A7 or hybridoma culture of Fancomaps and 950 [mu] Lt; / RTI > for 30 minutes. After washing, the procedure was repeated with 50 μl of rat-anti-mouse-IgM-antibody or rat-anti-mouse-IgG-antibody conjugated with microbeads (Miltenyi Biotec, KoIn, Germany). After washing, magnetically labeled TF-positive K562 cells were separated by two successive columns provided by Miltenyi Biotec (KoIn, Germany) as described in the manufacturer's manual. After 9 days of incubation, the separation procedure was repeated a total of 3 times. FACS analysis (flow cytometry) initiated with antibody staining: Approximately 3x10 ⁵ cells were incubated with primary monoclonal antibody (A78-G / A7 (IgM), Hybridoma culture supernatant of Pancomap (IgG1) All diluted 1: 2 in cell culture medium) and then incubated with secondary Cy3-conjugated goat anti-mouse IgM or IgG antibody diluted 1: 200 in PBS for 30 min at 4 [deg.] C and rewashed . Resuspended cells (200 [mu] l PBS) were examined by flow cytometry (flow cytometer: Coulter Epics, Beckman Coulter, Krefeld, Ger). For antibody-labeled cells, the following parameters: forward scatter (FS): 26 V, gain 1, sideward scatter (SS): 807 V, gain 5, FL2: 740 V, gain 1 , And quantitation was performed on the lectin-labeled cells using Expo32 software (Becton Coulter) with the following parameters: FS: 26 V, Gain 1, SS: 807 V, Gain 5, FL1: 740 V, .

After 3 rounds of separation, K562 cell clusters of 93% TF-positive cells were obtained. However, the proportion of TF-positive K562 cells decreases over time, reaching a bottom level of about 20% of TF-positive cells for a period of 14 days following the isolation procedure. For stable expression of the TF-positive phenotype, K562 cells were isolated for a later time and finally isolated TF-positive K562 cells were then cloned by limited dilution in 96-well plates (1 cell / 100 μl). Of the 30 K562 cell clones obtained, 17 cell clones did not express low amounts of TF antigen or TF antigen. These cell clones were analyzed by flow cytometry for SNA binding and for growth rate (analysis of doubling time, see below). Cell clones showing high SNA binding and high proliferation rates were selected. Stable NM-H9 clones were selected for further clonal development by single cell cloning and to optimize growth under serum-free conditions. Most preferably, the cell clone NM-H9D8 [DSM ACC2806] was selected and deposited with DSM ACC2806 ["DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH" in Braunschweig (Germany), by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (Germany) at the September 15, 2006].

Example  3

K562 , NM -F9, NM - D4 , NM - H9 , NM -E-2F9, NM -C-2F5, NM - H9D8 , NM - H9D8 - E6 , NM -H9D8-E6Q12, and GT -2x cell line and CHOdhfr - Cell culture and generation of serum-free cell line

H9D8-E6Q12 [DSM ACC2807], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12, NM-H9D8-E6Q12, NM- DSM ACC2856] ["DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH" in Braunschweig (Germany), by Glycotope GmbH, Robert-Rossle-Str. 10 or 13125 Berlin (Germany) on August 8, 2007] or GT-2x [DSM ACC] were cultured in RPMI 1640 or serum-free X-Vivo 20 medium supplemented with 10% FCS and 2 mM glutamine, Lt; RTI ID = 0.0 > 37 C. < / RTI >

CHOdhfr- cells (ATCC No. CRL-9096) were cultured in DMEM or serum-free CHO-S-SFM II medium supplemented with 10% FCS, 2 mM glutamine, and 2% HT supplement and cultured in 8% And grown at 37 ° C.

H9D8-E6Q12 [DSM ACC2807], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12, NM-H9D8-E6Q12, NM- DSM ACC2856], or GT-2x [DSM ACC] and cells or cell lines derived from the host cells are readily adapted to serum-free conditions by complete exchange of the cell culture medium. The cells and cell lines according to the present invention are inoculated into a serum-free medium such as X-Vivo 20 at a density of 1 x 10 ⁵ to 5 x 10 ⁵ cells / ml, preferably 2 x 10 ⁵ cells / ml, ≪ / RTI > After 4 to 7 days of culture, cells having reached a density of 5 x 10 ⁵ to 10 x 10 ⁵ cells / ml were selected as cells adapted to the serum-free medium. Adaptation to serum-free medium may be performed by serially diluting the medium supplemented with FCS with a serum-free medium composition (continuous adaptation). The productivity of the transformed FCS-producing clones of the antibody compositions producing the host cells of the present invention is largely conserved for serum-free conditions.

Adaptation of CHOdhfr- (ATCC No. CRL-9096) to serum-free conditions should be performed sequentially, taking several weeks and at least half of the productivity loss being common.

Example  4

In eukaryotic cells chimera  Antibody Pancomap , Panko  One, Panko  2 or Cetuxi Map  Of the expression vector Cloning

The variable sequence of the Vancomaps was PCR amplified from the murine hybridoma cells producing the Vancomaps using specific primers [H Cancer Immunol Immunother . H 2006 Nov; 55 (11): 1337-47. Epub 2006 Feb. PankoMab: a potent new generation anti-tumor MUC1 antibody. Daniel Czyk et al.

Variable sequences VH and VL of panco 1 and panco 2 are disclosed in WO2004 / 065423, which is incorporated herein by reference:

Variable heavy chain of Panco 1:

The variable light chain of Panco 1:

Variable heavy chain of Panco 2:

The variable light chain of Panco 2:

The variable amino acid sequence of Cetuxime maps VH and VL

로부터 수득하였고, 벡터NT1을 이용하여 cDNA 코딩 서열로 역번역하였다.

And reverse translated to the cDNA coding sequence using vector NTl.

Variable heavy chain of Cetuxime map:

Cetuximab's variable light chain:

The cDNA sequence was extended by Ncol / Nhel for VL and Ncol / Sall for VH to generate cDNA. The Ncol / Xhol-cut variable heavy chain fragment VH was cloned into an Ncol / Sall-cut BS-leader vector as described in WO2004 / 065423. The BS-leader vector comprises a cloning cassette for introducing a T cell receptor signal peptide sequence at the 5 'end of the variable domain and a splice donor sequence at the 3' end. The variable light chain VL of the corresponding antibody was amplified with a specific primer at the 3 'end which further encodes the splice donor site and cloned into a similarly digested BS-leader vector via Ncol / NheI. Each HindIII / BamHI fragment from the BS-leader vector was then cloned into a corresponding eukaryotic expression vector. This vector (pEFpuroC gamma 1VB _HB , pEFdhfrC kappa VB _LB , pEFdhfrB _mutB C kappa VB _LB ) contains the EF-1 alpha-promoter and HCMV enhancer, SV40 origin, polyadenylation signal and furomycin resistance gene in the vector for heavy chain (Dhfr) or K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-H9 and NM-E for selection and gene amplification for CHO cells H9D8-E6Q12 [DSM ACC2856], or GT-2x [DSM ACC] for the expression of SEQ ID NO: 1 (SEQ ID NO: 1 #), as well as the genomic sequence of human constant gamma 1 region for heavy chain or human constant kappa region for light chain (see WO 2004/065423 for primers and vector maps for amplification from genomic human DNA) .

Example  5

chimera  Antibodies, Pancomap , Panko  One, Panko  2 or Cetuxi Map  Of eukaryotic cells expressing Transfection  And a gene amplification procedure for producing high production cell clones in the presence of serum

NM-F9 [DSM ACC2606] or CHOdhfr- [ATCC No. CRL- 9096], in the case of suspension cells such as NM-F9, by lipofection using DMRIE-C or electroporation, or by lipofectamine or electroporation in the case of adherent cell line CHOdhfr- The cells were co-tracked with a mixture of the disclosed vectors (1: 1 to 1: 3) for the heavy and light chains. After two days of transfection, the growth medium was replaced with selective medium for one week (NM-F9 in RPMI 1640 + 10% FCS + 2 mM L-glutamine + 0.75 ug / ml puromycin + 50 nM methotrexate; CHOdhfr- in DMEM + 10% dialyzed FCS + 2 mM L-glutamine + 5 [mu] g / ml puromycin + 50 mM methotrexate). The first amplification was performed by increasing the methotrexate concentration to 100 nM for an additional two weeks. A portion of the amplified cell clusters were single cell cloned in the medium without the addition of puromycin and methotrexate and the rest of the cells were treated with a new round of gene amplification by increasing the methotrexate concentration. Four to six rounds of gene amplification (100, 200, 500, 1000, 2000, 3000 nM methotrexate) were performed in this manner. In addition, the most favorable production clones identified by clonal screening and analysis were further similarly amplified.

After single cell cloning in 96-well plates with limited dilution (0.5 cells per well), the cells were cultured for 2-3 weeks while analyzing the growing cell clones under a microscope, and the cloning efficiency in% Number of wells carrying clones x 100 / theoretical number of seeded cells). Growing clones were screened for productivity using different procedures for adherent CHOdhfr- cells or NM-F9 suspension cells.

CHOdhfr- : Growing clone cells were washed with PBS and harvested by accutase treatment. Half of resuspended cells were seeded in 96-well test plates, and the remaining half was seeded in 24-well plates for further incubation. The test plates were incubated for 20-24 hours. The supernatant of each well was analyzed for antibody titer as described in Determination of Specific Production Rate (SPR) and Doubling Time (g) (see below). Relative cell density was measured by MTT assay. Specifically, cells were incubated with MTT solution for 2 hours, the solution was discarded and the cells were lysed with 0.04 M HCl solution in 2-propanol. After 2 hours, the plates were mixed moderately and measured on a 630 nm reference filter in duplicate using a microtiter plate photometer equipped with a 570 nm filter.

NM-F9 : The 96-well plate was centrifuged and the supernatant was discarded. The cells were resuspended in 200 [mu] l of fresh medium. Half of resuspended cells were seeded in 96-well test plates and diluted to 100 쨉 l medium, and the remaining half was placed on a cloning plate for further incubation. Two days after incubation, the test plate was centrifuged and 20 ㎕ of supernatant was assayed for antibody titer as described in Determination of Specific Production Rate (SPR) and Doubling Time (g) (see below). Relative cell density was determined using WST-1 assays in which 10 [mu] l of WST-1 solution (Roche) was added in each well. One to three hours after incubation, measurements were made on a 630 nm reference filter in duplicate using a microtiter plate photometer equipped with a 450 nm filter.

Cell clones with a high ratio of antibody titer versus cell density were selected, cultured and further analyzed.

The conditions for K562, NM-D4 and NM-H9 were the same.

Specific production rate ( SPR ) And the doubling time (q)

For each clone, 2x10 < ⁴ > P cells were seeded per well of a 24-well tissue culture plate in 500 [mu] l growth medium. The cells were allowed to grow for 3 days, conditioned medium was collected for analysis, and cells were removed and counted, if necessary, by means of an acetase. Specific antibody titers were determined quantitatively from the media samples by ELISA. The assay plate was coated with human kappa chain specific antibody (BD). Bound recombinant antibodies were detected with an anti-human IgG (H + L) horseradish peroxidase (HRP) conjugate (Jackson Immunoresearch Laboratories). For quantification, purified recombinant chimeric antibodies were used as reference.

The SPR (picogram) of a particular protein measured per cell (pcd) is a function of both growth rate and productivity, and was calculated by the following equation:

The doubling time was calculated by the following equation:

g = log 2 x (incubation time) / log (final cell number / initial cell number)

Determination of average yield and maximum yield for cell lines

After single cell cloning from 200 theoretically plated monoclones in a 96-well plate with the disclosed limited dilution (0.5 cells per well), SPR was measured for growing cell clones and the mean and error were determined And determined the maximum yield for different cell lines and different conditions. Table 1 compares the data of chimeric vancomap-producing cell clones of CHOdhfr- and NM-F9 generated under serum-containing conditions.

Example  6

Generation of serum-free, high-yield cell clones producing antibody molecule compositions

The track selection of NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12 or GT-2x adapted to serum-free medium X-Vivo 20 was performed using DMRIE-C or electroporation (Nucleofector, Amaxa) Under serum-free conditions. After 2 days of transfection, the growth medium was exchanged for 1 week with selective medium (X-Vivo 20 + 0.75 [mu] g / ml puromycin + 50 nM methotrexate). The first amplification was performed by increasing the methotrexate concentration to 100 nM for an additional two weeks. A portion of the amplified cell clusters were cloned in a single cell in X-Vivo without the addition of puromycin and methotrexate and the rest of the cells were treated with a new round of gene amplification by increasing the methotrexate concentration. Four to six rounds of gene amplification (100, 200, 500, 1000, 2000, 3000 nM methotrexate) were performed in this manner. In addition, the most favorable production clones identified by clonal screening and analysis were further similarly amplified. Following single cell cloning in 96-well plates with limited dilution (0.5 cells per well), the plates were incubated for 2-3 weeks while analyzing the growing cell clones under a microscope and the cloning efficiency in% Number of wells carrying clones x 100 / theoretical number of seeded cells). Growing clones were screened for productivity in the presence of serum using the same procedure as NM-F9 suspension cells (see above).

Determination of average yield and maximum yield of cell clones

After single cell cloning from 200 theoretically plated monoclones in a 96-well plate with the disclosed limited dilution (0.5 cells per well), SPR was measured for growing cell clones and the mean and error were determined And the maximum yield for different conditions was determined. Table 2 presents data for chimeric pancomm-producing cell clones of NM-H9D8 [DSM ACC2806], completely generated under serum-free conditions.

The specific production rate (SPR) after amplification in the cell line according to the invention was higher than CHOdhfr- and the highest in chimeric fancomap-producing NM-H9D8 [DSM ACC2806] cells generated under serum-free conditions.

The production rate of most clones is very stable over at least 6 weeks without selective pressure. The best clone has a doubling time of 24 hours and is stable with an SPR of 30 pcd over 6 weeks.

The data reflects the productivity of the clones at the small-lab scale with additional possibilities for process development, medium optimization and increased productivity due to fermentation.

The higher productivity and yield of the resultant clones generated under serum-free conditions is desirable and the conditions are favorable for K562, NM-F9, NM-D4, NM-H9, NM- The same is true for all cell lines and all other antibodies such as C-2F5, NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856], or GT-2x [DSM ACC].

Example  7

Isolation of antibody molecule composition

For production and isolation of the antibody molecule composition according to the present invention, stable track-sputtered cells secreting chimeric antibodies Pancomap, Panco 1, Pancor 2 or Cetuximab are cultured in serum-free medium at a cell density of about 1 - And cultured until reaching 2 x 10P ^6P cells / ml. After removing the cells from the cell culture supernatant by centrifugation (400 xg, 15 min), the chimeric antibody was purified using protein A column (HiTrap r-protein A FF, Amersham Pharmacia Biotech). The purified antibody fractions eluted by sudden pH changes were re-buffered in PBS and concentrated using a Centriprep centrifuge tube (cut-off 50 kDa, Millipore)

Example  8

The antibody molecule composition according to the present invention Fc - Mediated  Cell cytotoxicity Determination of

From blood donors Effects  As a cell PBMC  detach

PBMC were isolated from the blood of healthy donors by density centrifugation using Ficoll-Hypaque (Biochrom). Cells were washed three times with RPMI 1640 supplemented with 5% FCS and kept at low temperature in a separate batch of 5x10 ⁷ cells. PBMCs were thawed and maintained overnight in RPMI 1640 (RPMI / FCS), either directly or supplemented with 10% FCS, and then used for flow cytometry or as effector cells in cytotoxicity assays.

In vitro  Detection of cytotoxicity of antibody-dependent cells in a model

Antibody-dependent cellular cytotoxicity (ADCC) of recombinant antibodies according to the invention was investigated by europium emission assay. The target cells (ZR-75-1, 5 x 10 ⁶ ) were incubated for 6 min on ice with 800 μl of europium buffer (50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , (710 V, 1 pulse, 30 μs) in a multipolyzer (Eppendorf) and incubated for an additional 6 minutes on ice Lt; / RTI > Cells were then washed five times in RPMI 1640/5% FCS and seeded into 96-well round bottom plates (Nunc; 1 x 10 ⁴ cells / 100 μl per well). After addition of 20 μl of recombinant antibody or corresponding control (medium, isotype control human IgG) at various concentrations (final concentration of 0.5-25 μg / ml in 200 μl incubation volume), human peripheral blood cells 80 [mu] l) was added as effector cells using an effector / target cell ratio of 50: 1. To determine spontaneous release, 80 [mu] l RPMI / FCS was added without effector cells. The maximum release was determined after complete dissolution of the target cells with ethanol. After incubation in the incubator at 37 ° C for 4 hours, the plates were centrifuged at 500 xg for 5 minutes and 25 μl of each sample was pipetted (Perkin-Elmer Wallac) to 200 μl per well of the enhancement solution . After incubation at room temperature for 15 minutes, fluorescence was measured (VictorP ^2P fluorometer, Perkin-Elmer Wallac). Specific cytotoxicity is obtained from the equation (experimental dissolution - spontaneous dissolution) / (maximal dissolution - spontaneous dissolution) * 100.

Alternatively, target cells (LS174T or ZR-75-1, 3 x ¹⁰⁶ cells) were incubated with 100 μl of europium buffer (50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 10 mM diethylenetriamine pentaacetic acid, 2 mM europium (III) acetate), electroporated in Nuclease Factor II (Amaxa) using Program A-011, And incubated for 6 minutes. Cells were then washed 4 times in RPMI 1640/5% FCS and seeded into 96-well round bottom plates (Nunc; 5-10 x 10 ³ cells / 100 μl per well). 20 [mu] l of recombinant antibody or equivalent of various concentrations (final concentration of 0.001 to 100 ng / ml in 200 [mu] l incubation volume for Cetuximab; 0.16 to 5 [mu] g / ml for chimeric fancomap, panco 1 or panco 2) Human peripheral blood cells (80 [mu] l per well) were added as effector cells using an effector / target cell ratio of 100: 1 to 50: 1 after addition of a control (medium, isotype control human IgG). 80 [mu] l of RPMI / FCS without effector cells was added to determine spontaneous release. The maximal release was determined after complete dissolution of the target cells with 1% Triton X-100. After incubation in the incubator at 37 ° C for 4 hours or overnight, the plates were centrifuged at 500 xg for 5 minutes and 25 μl of each sample was pipetted to 200 μl per well of the enhancer solution (Perkin-Elmer Wallac). After incubation at room temperature for 10 minutes, fluorescence was measured (Victor ² fluorometer, Perkin-Elmer Wallac). Specific cytotoxicity is obtained from the equation (experimental dissolution - spontaneous dissolution) / (maximal dissolution - spontaneous dissolution) * 100.

fut8 ^- ADCC activity of the city map setuk isolated from the cell lines NM-H9D8-E6 was significantly higher than the ADCC activity of the city map setuk CHOdhfr- isolated from the cells or SP2 / 0 cells. As shown in FIG. 2, the cetuximap isolated from NM-H9D8-E6 has an antibody concentration that is about 30-fold lower than the cetuximab isolated from CHOdhfr- or SP2 / 0 cells (from Erbitux, Merck) Lt; RTI ID = 0.0 > LS174T. ≪ / RTI > This shows 30-fold higher ADCC activity than commercial products. These results were obtained using the production method of the present invention resulting in an optimized glycosylation pattern.

DSM ACC2606], K562, NM-H9, NM-D4 [DSM ACC2605], NM-H9D8-E6 [DSM ACC2807], NM-H9D8- , Or NM-H9D8 [DSM ACC2806] is about 5-fold higher than the ADCC activity of chimeric fancomaps isolated from CHOdhfr- cells. Approximately the fifth chimeric pancomm concentration results in the same ADCC activity as the chimeric pancomm expressed in CHOdhfr- cells. Figure 3 shows individual results using the NM-F9 cell line for comparison purposes.

The ADCC activity of chimeric panco 1 isolated from NM-H9D8-E6 or NM-H9D8-E6Q12 is about 8-fold higher than the ADCC activity of chimeric panco 1 isolated from CHOdhfr- cells. Approximately the eighth chimeric panco 1 concentration results in the same ADCC activity as chimeric panco 1 expressed in CHOdhfr- cells. The individual results are shown in Fig.

The ADCC activity of chimeric panco 2 isolated from NM-H9D8 or GT-2x is about six times higher than the ADCC activity of chimeric panco 2 isolated from CHOdhfr- cells. Approximately the sixth chimeric panco2 concentration results in the same ADCC activity as chimeric panco 2 expressed in CHOdhfr- cells. The individual results are shown in Fig.

The ADCC activity of chimeric panco 2 isolated from NM-H9D8-E6 is lower than the ADCC activity of chimeric panco 2 isolated from NM-H9D8 cells. The individual results are shown in Fig.

In vitro  Determination of cytotoxicity of complement-sequence-dependent cells in the model

The cytotoxicity (CDC) of complement-sequence-dependent cells of the antibody was examined by europium emission assay. Eu ³⁺ -loaded target cells (ZR-75-1, 3 x 10 ⁶ ) were prepared as described above.

The cells were then seeded into 96-well round bottom plates (Nunc; 5 x 10 ³ cells / 100 μl per well). After addition of 20 μl of antibody or a corresponding control (medium, isotype control human IgG) at various concentrations (final concentration of 0.5-50 μg / ml in 200 μl incubation volume), the cells were incubated for 30 minutes at room temperature Respectively. Then, 10 [mu] l of rabbit complementary sequence (Cedarline, diluted 1: 5 to 1:10) per well was added. Spontaneous release was determined by the addition of 10 [mu] l of RPMI / FCS without complement. The maximum release was determined after complete dissolution of the target cells with ethanol or 1% Triton X-100. After incubation in the incubator at 37 占 폚 for 3 to 3.5 hours, the plates were centrifuged at 500 占 g for 5 minutes and 25 占 퐇 of each sample was pipetted to 200 占 퐇 per well of the enhancer solution (Perkin-Elmer Wallac). After incubation at room temperature for 10 minutes, fluorescence was measured (VictorP ^2P fluorometer, Perkin-Elmer Wallac). Specific cytotoxicity is obtained from the equation (experimental dissolution - spontaneous dissolution) / (maximal dissolution - spontaneous dissolution) * 100.

The CDC activity of chimeric fancomaps isolated from NM-F9 cells is eight times higher than the CDC activity of chimeric fancomaps isolated from CHOdhfr- cells. The same specific lysis as the chimeric pancomm isolated from CHOdhfr- cells is induced in chimeric pancomm isolated from NM-F9 at an 8-fold lower concentration. The individual results are shown in Fig.

To analyze the predatory activity of the antibody molecule composition Conjugate  Formation Test ( CFA )

PKH26 Tumor cell staining:

1x10P ^7P to 2x10P ^7P tumor cells were washed twice in PBS, resuspended in 1 ml of diluent C and 1 ml of PKH26 was added to ZR-74-1, ZR-74-1TF, MCF-7 and MT-3 At a concentration of 12 x 10P- ^6PM .

After 3 minutes incubation at room temperature, staining was stopped by adding 2 ml FCS for 1 minute at room temperature. The medium (4 ml, RPMI supplemented with 1% L-glutamine, 10% FCS) was added, and the cells were spun down and washed four times with medium. Tumor cells were incubated at 37 ° C, 5% COB _2B to release excess PKH-26.

Optimization of PKH-26 staining was performed using half the cell volume and volume.

CFA :

PKH-26 labeled tumor cells were harvested with trypsin / EDTA, washed once with PBS and resuspended in MAK cell medium (Invitrogen) in the presence or absence of recombinant antibody molecule composition at 0.8 x 10P ^6P cells / ml. Tumor cells (250 쨉 l, 0.2 x 10P ^6P cells) were seeded in non-adherent polypropylene tubes.

Boyer et al., Exp. Hematol. 27, 751-761 (1999)), washed and resuspended at 1.6 x 10P ^6P cells / ml. 250 [mu] l of MAK cells (0.2 x 10P ^6P cells) were added to PKH-26 labeled tumor cells (E: T ratio 2: 1). Individual samples were incubated in duplicate at 4 ° C and 37 ° C / 5% COB _2B for 3 hours or on (18-2Oh).

After incubation at 3 h or o.n., the cells were washed with PBS and stained with CD11c-FITC (1: 18,5) + 7-AAD (1: 500) in PBS / 10% FCS. Cells were washed with PBS and resuspended in 400 [mu] l PBS. Acquisition was performed on Epics XL (Beckman Coulter) on 10.000 cells. The proportion of colonized MAK cells was determined as the proportion of PKH-26 positive cells in gated cell clusters for all viable CD11c-FITC positive cells.

Example  9

ELISA Of binding activity of antibody molecule composition

To analyze the antigen binding of antibody molecule compositions expressed in different cell lines, purified antibody molecule compositions of chimeric pancomaps and panco 2 were prepared by ELISA on synthetic glycosylated MUC1 30-mer peptides carrying the sequence APPAHGVTSAPDT [GalNAc alpha] RPAPGSTAPPAHGVTSA Respectively. Non-glycosylated MUC1 peptide was used as a control.

As part of the -20 ℃ using the stored stock solution (HB _2B O 1 of the 1 ml bidest mg.), It gave the dilution of 1 ㎍ / ml in PBS. 50 [mu] l / well was pipetted on a NUNC immune plate F96 MaxiSorp and the test plate was incubated overnight at 4 [deg.] C. The next day, the test plate was washed three times with PBS / 0.2% Tween-20. Subsequently, non-specific binding sites were blocked with 2% BSA in PBS, incubated at room temperature for 1 hour or more, and 50 [mu] l of recombinant doCoMaM applied (1-400 ng / ml PBS / 1% BSA). After three washing steps with PBS / 0.2% Tween-20, a peroxidase-coupled secondary anti-human Fc [gamma] l antibody was applied to detect specifically bound antibodies. To detect the bound secondary antibody, a color reaction using TMB (3,3 ', 5,5'-tetramethylbenzidine) was performed. After 15 minutes, the reaction was quenched by the addition of 2.5 N HB ₂ SOB _4B . Measurements were made on a 630 nm reference filter in duplicate using a microtiter plate photometer equipped with a 450 nm filter.

The binding activity of chimeric fancomaps isolated from NM-F9 is about 50% higher than the binding activity of chimeric fancomaps isolated from CHOdhfr- cells (Figure 8).

The binding activity of isolated chimeric panco 2 expressed and isolated from high sialylated clone NM-H9D8 in ELISA is comparable to that of GT-2x or higher than that of CHOdhfr- (Figure 9).

Example  10

NM -F9, NM - and CHOdhfr Of the antibody molecule composition expressed in the cell Glycann  analysis

The glycans of at least 100 [mu] g of the purified antibody were cleaved by acid hydrolysis. Sialic acid was specifically labeled by conjugation with fluorescent dye DMB. The analysis was carried out, for example, by HPLC on an Asahipak-NH2 column to isolate the saccharide. Identification and yielding of sialic acid was carried out by means of the appropriate standard sialic acid.

Analysis of the chimeric Fancomab antibody molecule composition expressed in CHOdhfr- or NM-F9 cells showed <10% monosialylation of the CHOdhfr-product and almost no sialylation of the NM-F9 or GT-2x product. The latter contained only non-charged glycans.

More specifically, the glycans of at least 100 [mu] g of the purified antibody were cleaved by acid hydrolysis. Sialic acid was specifically labeled by conjugation with a fluorescent dye such as DMB. Analysis was carried out, for example, by HPLC on reversed phase chromatography on RP-18 column. Identification and yielding of sialic acid was carried out by means of the appropriate standard sialic acid.

To determine the charged glycan structure and galactosylation state, 100 [mu] g of antibody was digested with trypsin and N-glycans were obtained by treatment with PNGaseF. The purified glycans were labeled with 2-aminobenzamide (2-AB) and treated with anion exchange chromatography HPLC (Asahi-PAK-column) for determination of the charged N-glycan structure. For the determination of the galactosylation state, the N-glycans were treated with neuraminidase for depletion of sialic acid content, separated by amino phase HPLC (Luna-NH2-column) and the peaks were analyzed by mass spectroscopy Lt; / RTI &gt;

Peaks containing potential bisecting GIcNAc carrying glycans were separated in a second step by reverse phase chromatography (RP18-column). By this, it is possible to distinguish the triantennary and the biantennary + bisecting structure. The degree of fucosylation of the antibody was determined by amino-phase HPLC (Luna-NH2-column) of glycan with 2-AB labeling. The peak was quantified by integration and the underlying glycan structure was analyzed by mass spectrometry. The fucosylated and fucosylated structure was determined and the integrated peak area was determined.

The different antibody molecule compositions of Cetuximab, Pancomaps and Panco 1 expressed in NM-F9, GT-2x, NM-H9D8, NM-H9D8-E6 or CHOdhfr- cells were analyzed and the results are shown in Tables 3-4 Respectively.

Antibody sialylation was characterized by ELISA or Western blot analysis using a lectin preferentially bound to alpha 2-6 (SNA) or alpha 2-3 (MAL-I) bound sialic acid.

Western blot analysis was performed to identify differently sialylated heavy chains of antibody molecule compositions expressed in CHOdhfr-, NM-F9, or NM-H9D8 [DSM ACC2806]. 5 μg of each antibody molecule composition was separated under reducing conditions in 10% acrylamide gel by SDS-PAGE. Proteins were transferred to nitrocellulose and visualized by lectin and / or secondary anti-human IgG antibodies. Figure 10 is a graph showing the effect of a second anti-human IgG antibody (Figure 10A) or SNA (Figure 10B) detecting 2-6 sialylation in CHOdhfr-, NM-F9, or NM-H9D8 [DSM ACC2806] Lt; RTI ID = 0.0 &gt; of &lt; / RTI &gt; antibody molecule composition. Figure 11 shows the differently sialylated heavy chains of cetuximab antibody molecule compositions expressed in CHOdhfr- or NM-H9D8 visualized by SNA binding.

ELISA experiments were used to analyze the sialylation of antibodies isolated from the supernatants of NM-F9, NM-H9D8, or NM-H9D8-E6 cells. 2 μg / ml in PBS and 50 μl per well of purified antibody were coated overnight at 4 ° C with 96 well microtiter plates (Maxisorp), blocked (PBS / BSA), washed and incubated with biotinylated SNA (2 Mu] g / ml, PBS / 1% BSA) for 1 hour and washed again. The wells were then incubated with POD-streptavidin, washed, and generated with TMB. The reaction was stopped by adding 2.5NH ₂ SO ₄ and optical density was measured at 630 nm as reference at 450 nm. Figure 12 shows that the binding of SNA generates antibodies expressed in NM-H9D8 or NM-H9D8-E6 cells, but does not generate antibodies expressed in CHOdhfr- or GT-2x cells. The intensity of the binding is different for the availability of the saccharide under the conditions of the individual antibody or conditions used, indicating different degree of sialylation caused by the microstructure of the individual antibody.

ELISA experiments were performed by coating purified antibodies expressed in different cell lines in wells of microtiter plates (2 ug / ml, well per well) and detecting suitable biotinylated lectin SNA and MAL I. The dependence of lectin binding by sialylation was examined by neuraminidase treatment (incubation at 0.1 U / ml and room temperature for 1 hour). The results for the Cetuxim map are illustrated in Figures 13A and 13B.

Dot blot analysis was performed to identify differences in sialylation of antibodies expressed in CHOdhfr-, NM-F9, NM-H9D8, and NM-H9D8-E6 cells. 3 [mu] g of the purified antibody was spotted on nitrocellulose, blocked with PBS / BSA, washed, and visualized by SNA detecting 2,6-sialylation. Figure 14 shows blots of chimeric antibody panco 1 and panco 2 isolated from CHOdhfr-, NM-F9, NM-H9D8, or NM-H9D8-E6 cells. 2,6-sialylation can only be detected on antibodies isolated from NM-H9D8 or NM-H9D8-E6 cells.

Example  11

NM -F9, NM - H9D8  And CHOdhfr - Detection of Bioavailability of Antibody Molecule Composition Expressed in Cells

Longer bioavailability was measured in the sialylated antibody levocomap expressed in NM-H9D8 compared to the un-sialylated antibody levocomplex expressed in NM-F9 cells in nude mice. 5 [mu] g of purified antibody per mouse was administered to at least 3 mice per group i.v. Respectively. Blood samples were collected at different time points after injection (5 minutes, 2, 5, 8, 24, 48, 72, and 144 hours after injection), sera were separated and samples were stored at -80 ° C until analysis. Antibody titers were determined by ELISA in these samples. Figure 15 shows that chimeric pancomm isolated from NM-H9D8 cells can be used longer than those isolated from NM-F9 cells, which is probably caused by different sialylation of antibodies.

Example  12

In eukaryotic cells hFSH Of the vector expressing Cloning

The coding sequences of the FSH alpha and FSH beta chains were PCR amplified with specific primers using the BAC clones RZPDB737B122053D (FSH alpha genome), IRAUp969E0752D (FSH alpha cDNA) and RZPDB737H0619D6 (FSH beta genome) obtained from RZPD (Germany).

FSH Alpha Chain Primer:

FSH Beta Chain Primer:

After amplification, the product was cloned into the corresponding eukaryotic expression vector via Hindlll / BamHI (FSH genomic beta) and Kpnl / BamHI (FSH genomic alpha or FSH cDNA alpha). The cDNA sequence was generated by gene synthesis encoding the FSH beta chain fused to the TCR-leader sequence and cloned into a eukaryotic expression vector (FSHcDNA beta).

Sequence FSHcDNA beta (TCR-leader sequence underlined):

Expression vectors (pEFpuro and pEFdhfrB _mutB ) contained EF-1 alpha-promoter and HCMV enhancer, SV40 origin, polyadenylation signal, and furomycin resistance gene in a vector for alpha chain, and murine dihydroporazole 1 (SEQ ID NO: 1) for expression of NM-F9, GT-2x, K562, NM-H9, NM-D4 or NM-H9D8 for gene (dhfr) .

Example  13

Human recombination hFSH Of eukaryotic cells expressing Transfection  And Serum-free  Conditions (GT-2x and NM - H9D8 ) Or serum containing conditions ( CHOdhfr -) &lt; / RTI &gt; amplification procedure to generate high producing cell clones

GT-2x (FSH cDNA alpha / FSH genome beta) and NM-H9D8 (FSH cDNA (SEQ ID NO: 2)) were used for expression of hFSH in GT-2x [DSM ACC], NM-H9D8 (DSM ACC2806) and CHOdhfr- (ATCC No. CRL- Alpha] / FSHcDNA beta), or by lipofection using DMRIE-C or electroporation (Nucleorefector; Amaxa) or in the case of the adherent cell line CHOdhfr- (FSH genome alpha / FSH genomic beta) The cells were co-tracked with a mixture of the disclosed vectors (1: 1 to 1: 3) for the alpha and beta chains by electroporation. This is an exemplary combination used in this step setup. Different combinations of each of the hFSH alpha and hFSH beta chain expression plasmids result in comparable results.

After two days of transfection, the growth medium was replaced with selective medium for one week (GT-2x and NM-H9D8NM-F9 in X-Vivo20 medium + 0.75 占 퐂 / ml puromycin + 50 nM methotrexate; CHOdhfr- in DMEM + 10% dialyzed FCS + 2 mM L-glutamine + 5 [mu] g / ml furomycin + 50 mM methotrexate). The first amplification was performed by increasing the methotrexate concentration to 100 nM for an additional two weeks. Some of the amplified cell clusters were single cell cloned in the medium without the addition of puromycin and methotrexate. The rest of the cells were treated with a new round of gene amplification by increasing the methotrexate concentration. Two to three rounds of gene amplification (100, 200, 500 nM methotrexate) were performed in this manner. In addition, the most favorable production clones identified by clonal screening and analysis were further similarly amplified.

CHOdhfr- : Growing clone cells were washed with PBS and harvested by treatment with acutase . Half of resuspended cells were seeded in 96-well test plates, and the remaining half was seeded in 24-well plates for further incubation. The test plates were incubated for 20-24 hours. The supernatant of each well was analyzed for antibody titer as described in Determination of Specific Production Rate (SPR) and Doubling Time (g) (see below). Relative cell density was measured by MTT assay. Specifically, cells were incubated with MTT solution for 2 hours, the solution was discarded and the cells were lysed with 0.04M HCl solution in 2-propanol. After 2 hours, the plates were mixed moderately and measured on a 630 nm reference filter in duplicate using a microtiter plate photometer equipped with a 570 nm filter.

GT-2x and NM-H9D8 : The 96-well plate was centrifuged and the supernatant discarded. The cells were resuspended in 200 [mu] l of fresh medium. Half of resuspended cells were seeded in 96-well test plates and diluted to 100 쨉 l medium, and the remaining half was placed on a cloning plate for further incubation. Two days after incubation, the test plate was centrifuged and 20 ㎕ of supernatant was assayed for antibody titer as described in Determination of Specific Production Rate (SPR) and Doubling Time (g) (see below). Relative cell density was determined using WST-1 assays in which 10 [mu] l of WST-1 solution (Roche) was added in each well. One to three hours after incubation, measurements were made on a 630 nm reference filter in duplicate using a microtiter plate photometer equipped with a 450 nm filter.

Specific production rate ( SPR ) And the doubling time (g)

For each clone, 2x10 &lt; ⁴ &gt; P cells were seeded per well of a 24-well tissue culture plate in 500 [mu] l growth medium. The cells were allowed to grow for 3 days, conditioned medium was collected for analysis, and cells were removed and counted, if necessary, by means of an acetase. Specific antibody titers were determined quantitatively from the media samples by ELISA. The assay plate was coated with mouse mAB specific for hFSH beta (ab22473). The recombinant recombinant hFSH was incubated with a chlorine polyclonal antibody specific for hCG alpha (ab20712) and detected with donkey anti-chlorine IgG H + L-POD (Jackson lmmuno Cat. No. 305-035-003). For quantification, purified recombinant hFSH was used as a reference.

The SPR (picogram) of a particular protein measured per cell (pcd) is a function of both growth rate and productivity, and was calculated by the following equation:

The doubling time was calculated by the following equation:

g = log 2 x (incubation time) / log (final cell number / initial cell number)

Determination of average yield and maximum yield for cell lines

After single cell cloning from 200 theoretically plated monoclones in a 96-well plate with the disclosed limited dilution (0.5 cells per well), SPR was measured for growing cell clones and the mean and error were determined And determined the maximum yield for different cell lines and different conditions. Table 7 and Table 8 compare the data of cell clones for the generation of hFSH of CHOdhfr-, GT-2x and NM-H9D8 produced under serum-containing conditions (CHOdhfr-) and serum-free conditions (GT-2x and NM-H9D8) do.

Example  14

hFSH  Isolation of molecular composition

For the generation and isolation of hFSH molecule composition of the present invention, culturing the illustration stably track specification cells secreting hFSH in serum-free medium, the cells until the density reaches about 1 to 2 x 10P ^6P cells / ml After removing the cells from the cell culture supernatant by centrifugation (400 xg, 15 min), the chimeric antibody was reacted with polyclonal antibody against hCG alpha coupled to NHS activated column (HiTrap NHS-activated HP; GE Healthcare) The purified FSH fractions eluted by sudden pH changes were re-buffered in PBS and concentrated using a Centrifuge centrifuge tube (cut-off 10 kDa, Millipore) , And analyzed by SDS-PAGE (Fig. 16).

Example  15

According to the invention FSH  Determination of the activity of the molecular composition

The activity of FSH molecules having different glycosylation patterns can be determined following the disclosed method. To select a suitable cell line that provides optimized glycosylation with the screening method according to the present invention, FSH molecules can be expressed in different cell lines FSH compositions were obtained from individual cell lines exhibiting different glycosylation patterns with respect to degree of sialylation, galactosylation and / or fucosylation. The FSH molecules / compositions with the most favorable activity and thus the glycosylation pattern were obtained by the following method &Lt; / RTI &gt; can be determined by one or more of:

Rat Granular layer  Cell assay:

Granule cells were obtained from DES-treated pituitary-resected rats. Methods for the preparation of cells are described in detail in Jia and Hsueh 1985.

The obtained cells were treated with 10O nM DES, 0,125 M 1-methyl-3-isobutylxanthine, 2 mM glutamine, 1 μM androstendione, 100 U / ml penicillin and 100 μg / cultured in McCoy's 5A medium containing increasing concentrations of rFSH product derived from 1 μg / ml streptomycin, 1 μg / ml bovine insulin and GT-2x and NM-H9D8 cells (0-1 μg / ml) The medium was collected 72 hours after incubation and stored at -20 ° C until assayed by ELISA (Calbiotech # ES071S).

293 HEK  black:

HEK293 cells stably transfected with hFSH-receptor (2xlO &lt; ⁵ &gt; cells / 35mm culture dish) were cultured in the presence of 0.125 mM 1-methyl-3-isobutylxanthin in the range of 0-1 / / ml FSH for 24 hours (Intracellular- and extracellular) cAMP concentrations were measured in cAMP direct biotrack (&lt; RTI ID = 0.0 &gt; Biotrak) EIA (GE Healthcare, Cat. No. RPN225) according to the manufacturer's instructions.

GFSHR -17 cell assay:

Cells 5% fetal calf serum (Biochrom KG, Berlin, Germany) in Dulbecco's modified Eagle's medium Ham F12 containing; were incubated with (1:: 1 v v Biochrom KG, Berlin, Germany) cells 2x10 ⁵ cells / Plates were plated in 24-well plates and incubated with GT-2x and NM-H9D8 cells for 24 hours with FSH protein molecule compositions obtained in the range of 0-1 ㎍ / ml for 1-24 hours. After incubation, the medium was collected and treated with progesterone And stored at-20 C until assayed for progesterone using an ELISA assay (Biochem Immunosystems, Freiburg, Germany) according to the manufacturer's instructions.

human Granular layer  Cell assay

Granulocyte-Lutein cells from follicular aspirates obtained from women participating in the in vitro fertilization program of the University of Stuttgart were hatched as described in Khan et al., 1990 and seeded with 6-well plates (1 per well -1.5 x 10 ⁵ cells grow in number) in a 37 ℃ 10% open cell-inactivated fetal calf serum (FCS; Gibco, Eggenstein, Germany ), penicillin (50 units / ml), streptomycin (50 pg / ml 5% in HAM's F12 / dulbecco modified essential medium (DMEM) (1: 1; ICN Biomedicals, Meckenheim, Germany) supplemented with gentamicin (100 pg / ml) and amphotericin CO ₂ atmosphere. Cells were incubated with increasing concentrations of FSH from GT-2x and NM-H9D8 cells for 24 hours in the range of 0-1 / / ml FSH in the presence of 0.125 mM 1-methyl-3-isobutylxanthin After incubation, total (intracellular- and cell-free) cells were incubated at &lt; RTI ID = 0.0 &gt; ) It was determined according to the manufacturer's instructions by the cAMP concentration in the direct cAMP EIA bio Track ™ (GE Healthcare, Cat.no .: RPN225).

Example  16

GT -2x and CHOdhfr - expressed in cells FSH  Of the composition Glycann  analysis

Western blot analysis was performed to identify FSH molecular compositions that were sialylated differently in CHOdhfr-, GT-2x [DSM ACC], or NM-H9D8 [DSM ACC2806]. 5 μg of each antibody molecule composition was analyzed by SDS-PAGE Protein was transferred to nitrocellulose and visualized by SNA to detect 2-6 sialylation (Figure 17).

Detailed glycan analysis can be performed according to Example 10 to determine sialic acid content, charge distribution, fucose content, galactosylation status and bisecting GlcNAc content.

This result shows that FSH can be produced with a very high yield in GT-2x (see FIG. 17). Here, the product also did not show any detectable 2-6 linked NeuNAc 1) Conversely, the FSH produced in NM-H9D8 exhibited a detectable somewhat higher amount of 2-6 bound NeuNAc. Activity can be detected by the method described in Example 15.

IUPAC - IUBMB  Definitions as recommended (1996):

Neu5Gc : 5-N- glycolyl - α -neuraminic acid

Neu5Ac and NeuNAc : 5-N-acetyl-alpha-neuraminic acid

Gala 1,3 Gal and Gal alpha 1,3 Gal: α-D- galacto-pyrazol nosil - (1 → 3) - galactosidase nosil pyrazol-adjacent saccharide

2,3 linked neuraminic acid: 5-N-acetyl -? - neuraminyl- (2? 3) - adjacent saccharides

2,6 linked neuraminic acid: 5-N-acetyl -? - neuraminyl- (2 → 6) -negative saccharide

It is to be understood that the present invention is not limited to these specific methods, protocols, and / or reagents disclosed herein, although the terminology used herein is for the purpose of describing particular examples only and is to be limited only by the scope of the appended claims. And is not intended to limit the scope of the invention.

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- The second half GlcNAc
- High yield
- 6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- high galactose as defined in paragraph 2 and the dependent claims
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- Higher affinity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- the low fucose as defined in paragraph 2 and the dependent claims;
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- The second half GlcNAc
- High yield
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High activity, especially Fc activity
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High activity, especially Fc activity
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High activity, especially Fc activity
- 2-6 NeuNAc
- No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High yield
- 2-6 NeuNAc
- 2-3 NeuNAc
- more sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High yield
- the minor sialic acid as defined in paragraph 2 and the dependent claims - No NeuGc
- Detectable terminal Gal alpha 1-3Gal None
- High yield
- 2-6 NeuNAc

Table 10

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2606 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2605 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2806 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2807 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2856 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

Reference number: 48 377 K

Access number DSM Additional indication according to PCT / RO / 134 format for ACC2858 :

Applicants are requested to ensure that the provision of samples of deposited biological materials referred to in this application can be used exclusively by independent named experts for countries having individual provisions (requests for "specialized solutions" to be applied, , Canada, Croatia, Denmark, Finland, Germany, Iceland, Norway, Singapore, Spain, Sweden, United Kingdom, Europe).

Accordingly, in the case of Europe, Applicants agree that, as provided in Rule 28 (3) EPC for a period of twenty (20) years from the date of filing, if a sample of the deposited biological material is considered to have been rejected, (Rule 28 (4) EPC) to be appointed by the person requesting the sample by payment of the sample.

                         SEQUENCE LISTING <110> Glycotope GmbH   <120> Fully human high yield production system for improved antibodies        and proteins <130> 48 377 <140> PCT / EP2007 / 007877 <141> 2007-09-10 <160> 21 <170> PatentIn version 3.3 <210> 1 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 1 Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asp Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Trp             20 25 30 Lys Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp                 85 90 95 Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Gln         115 120 125 Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp             180 185 <210> 2 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 2 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Ser             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 3 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 3 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Ala             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 4 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 4 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Gly             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 5 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 5 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Tyr Pro Trp Pro Pro Leu Arg Asn Glu Phe             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 6 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 6 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg Asn Glu Phe             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 7 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 7 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Phe Pro Trp Pro Pro Leu Arg Asn Glu Phe             20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp                 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His         115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Glu Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp             180 185 <210> 8 <211> 187 <212> PRT <213> Unknown <220> <223> MTX resistant DHFR variant <400> 8 Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg Asn Glu Phe             20 25 30 Lys Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp                 85 90 95 Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Gln         115 120 125 Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp             180 185 <210> 9 <211> 187 <212> PRT <213> Unknown <220> <223> MTX-resistant DHFR variant <400> 9 Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Tyr Pro Trp Pro Pro Leu Arg Asn Glu Phe             20 25 30 Lys Tyr Phe Gln Arg Met Thr Thr Ser Ser Val Glu Gly Lys Gln         35 40 45 Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys     50 55 60 Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp                 85 90 95 Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp Met             100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Gln         115 120 125 Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Glu     130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ile                 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp             180 185 <210> 10 <211> 18 <212> PRT <213> Unknown <220> <223> secretion peptide signal <400> 10 Met Trp Leu Gly Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala          <210> 11 <211> 117 <212> PRT <213> Unknown <220> <223> variable heavy chain of the antibody Panko -1 <400> 11 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala             20 25 30 Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Val Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr                 85 90 95 Tyr Cys Thr Arg Gly Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Thr Leu Thr Val Ser         115 <210> 12 <211> 113 <212> PRT <213> Unknown <220> <223> variable light chain of the antibody Panko 1 <400> 12 Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Glu Leu Lys             100 105 110 Arg      <210> 13 <211> 116 <212> PRT <213> Unknown <220> <223> variable heavy chain of the antibody Panko 2 <400> 13 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr             20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala Glu     50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Ser Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile Tyr                 85 90 95 Tyr Cys Thr Arg His Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr             100 105 110 Leu Thr Val Ser         115 <210> 14 <211> 113 <212> PRT <213> Unknown <220> <223> variable light chain of the antibody Panko 2 <400> 14 Asp Ile Val Met Thr Gln Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Ser Ser Leu Leu His Ser             20 25 30 Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly Leu Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro     50 55 60 Asp Arg Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn                 85 90 95 Leu Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 15 <211> 118 <212> PRT <213> Unknown <220> <223> variable heavy chain of the antibody Cetuximab <400> 15 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr             20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu         35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr     50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala                 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser         115 <210> 16 <211> 108 <212> PRT <213> Unknown <220> <223> variable light chain of the antibody Cetuximab <400> 16 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn             20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr                 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg             100 105 <210> 17 <211> 31 <212> DNA <213> Artificial <220> <223> Primer for FSH alpha chain <400> 17 aaaggtacca tggattacta cagaaaatat g 31 <210> 18 <211> 29 <212> DNA <213> artificial <220> <223> primer for FSH alpha chain <400> 18 aaaggatcct taagatttgt gataataac 29 <210> 19 <211> 30 <212> DNA <213> Artificial <220> <223> Primer for FSH beta chain <400> 19 tttaagctta tgaagacact ccagtttttc 30 <210> 20 <211> 27 <212> DNA <213> Artificial <220> <223> primer for FSH beta chain <400> 20 tttggatcct tattctttca tttcacc 27 <210> 21 <211> 399 <212> DNA <213> Artificial <220> <223> cDNA sequence <220> <221> misc_feature <222> (1) (63) <223> TCR leader sequence <400> 21 atggcctgcc ccggcttcct gtgggccctg gtgatcagca cctgcctgga attctccatg 60 gctaacagct gcgagctgac caacatcacc atcgccatcg agaaagagga atgccggttc 120 tgcatcagca tcaacaccac ctggtgcgcc ggctactgct acacccggga cctggtgtac 180 aaggaccccg ccaggcccaa gatccagaaa acctgcacct tcaaagaact ggtgtacgag 240 accgtgcggg tgcccggctg cgcccaccac gccgacagcc tgtacaccta ccccgtggcc 300 acccagtgcc actgcggcaa gtgcgacagc gacagcaccg actgcaccgt gaggggcctg 360 ggccccagct actgcagctt cggcgagatg aaagagtga 399

Claims (47)

A method for producing a protein molecular composition comprising: (a) introducing one or more nucleic acids encoding at least a portion of the protein into host cells, immortalized human blood cells derived from cell line K562 [ATCC CCL-243]; (b) culturing the host cell under conditions that allow the production of the protein molecular composition; And (c) separating the protein molecular composition, The host cell is selected to produce a protein molecular composition having the following glycosylation characteristics: NM-H9D8 [DSM ACC2806], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856] And GT-2X [DSM ACC2858] and cells or cell lines derived therefrom. (i) does not include detectable NeuGc; (ii) alpha 2-6 bound NeuNAc; And  (iii) Expression of CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 15% higher. The method according to claim 1, Wherein said host cell is selected to produce a protein molecular composition having one or more of the following characteristics: (i) does not contain detectable terminal Gal alpha 1-3Gal; (ii) at least 2% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iii) at least 5% carbohydrate structure of at least one particular carbohydrate chain or an entire carbohydrate unit at a specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iv) CHOdhfr- [ATCC No. Compared to the same amount of protein molecules in one or more protein molecular compositions of the same protein molecule isolated from the same protein molecule isolated from a protein carbohydrate structure or a specific glycosylation site of a protein molecule of the protein molecule composition, Structure, an increased degree of sialylation with a NeuNAc amount of at least 20% greater; (v) protein molecules of the protein molecular composition lacking fucose include at least 50% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glycosylation site of the white matter molecule; And (DSM ACC2606] or NM-D4 [DSM ACC2605] cells have higher degree of sialylation than those obtained in (vi) NM-F9 [DSM ACC2606] or NM- But only 50 to 60% of the degree of sialylation obtained for the host cells. The composition of claim 1, wherein the host cell is selected to produce a protein composition having one or more of the following glycosylation characteristics, wherein the protein molecule has at least one of the following glycosylation characteristics (i) - (v) How to create: (i) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, CHOdhfr- [ATCC No. Having an increased degree of galactosylation as compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from CRL-9096; (ii) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096] at least 5% higher in the amount of G2 structure compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from the same protein molecule; (iii) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096], at least 5% lower than the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from the same protein molecule; (iv) the total carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. Comprising at least 5% lower amount of fucose as compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from CRL-9096; And (v) Expression CHOdhfr- [ATCC No. Has a modified sialylation pattern relative to the sialylation pattern of one or more protein molecular compositions of the same protein molecule isolated from &lt; RTI ID = 0.0 &gt; CRL-9096. 4. The method of claim 3, wherein the sialylated pattern is characterized by one or more of the following features: At the time of expression, CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 20% higher than that of the native structure; And At the time of expression, CHOdhfr- [ATCC No. In a specific glycosylation site of the protein molecule of the protein molecule composition, as compared to the same amount of the protein molecule in one or more protein molecule compositions of the same protein molecule isolated from the same protein molecule isolated from the same protein molecule, And carbohydrate chains bound by charged N-glycosides in excess of at least 20% of the carbohydrate units. 2. The method of claim 1, wherein the host cell comprises one or more of the following features and is selected to produce a glycoprotein: At least one particular carbohydrate chain or at least 10% of the carbohydrate structure of the entire carbohydrate unit at a particular glycosylation site of the protein molecule in the protein molecules of the protein molecule composition lacking fucose;  At least one particular carbohydrate chain or at least 5% carbohydrate structure of the entire carbohydrate unit at a specific glycosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; A G2 structure in excess of 35% on a carbohydrate structure or a total carbohydrate structure at one particular glycosylation site of the protein molecule of the protein molecule composition; And Or less than 22% of the GO structure on a carbohydrate structure or a total carbohydrate structure at a particular glycosylation site of the protein molecule of the protein molecular composition. The method of claim 1, wherein the resulting protein molecular composition comprises one or more of the following features: The cell line CHOdhfr- [ATCC No. CRL-9096], has increased activity and increased yield relative to one or more protein molecular compositions of the same protein molecule; The cell line CHOdhfr- [ATCC No. CRL-9096], has improved homogeneity compared to one or more protein molecular compositions of the same protein molecule; The cell line CHOdhfr- [ATCC No. CRL-9096] that is at least 10% higher than the yield of one or more protein molecule compositions from the same protein molecule upon expression in the same protein; The cell line CHOdhfr- [ATCC No. &Lt; / RTI &gt; CRL-9096] as compared to one or more protein molecular compositions of the same protein molecule; The cell line CHOdhfr- [ATCC No. CRL-9096] as compared to one or more protein molecule compositions of the same protein molecule; When the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. Nos. Has a cytotoxicity of increased Fc-mediated cell that is at least 2-fold higher than the cytotoxicity of Fc-mediated cells of one or more antibody molecule compositions from the same antibody molecule upon expression in CRL-9096; And When the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. Nos. CRL-9096] has at least 15% higher antigen-mediated binding or Fc-mediated binding than binding of one or more antibody molecule compositions from the same antibody molecule. The method of claim 1, wherein the host cell is selected to produce a protein composition comprising a protein molecule having a glycosylation combination characterized by: (a) does not contain detectable NeuGc Does not contain detectable Gal alpha 1-3 Gal Comprising a galactosylation pattern as defined in claim 3 Has a fucose content as defined in claim 3 Includes bisecting GlcNAc The cell line CHOdhfr- [ATCC No. CRL-9096], or an increased amount of sialic acid compared to a sialylated deficient cell line compared to the protein composition of the same protein molecule; (b) does not contain detectable NeuGc Does not contain detectable Gal alpha 1-3 Gal Comprising a galactosylation pattern as defined in claim 3 Has a fucose content as defined in claim 3 Includes bisecting GlcNAc 2-6 NeuNAc. The method of claim 1, wherein the cell or cell strain is selected from the group consisting of NM-H9D8 [DSM ACC 2806], NM-H9D8-E6 [DSM ACC 2807], NM-H9D8-E6Q12 [DSM ACC 2856] Wherein the host cell is used. 4. The method of claim 1, wherein the nucleic acid is introduced into a host cell to encode an antifolate resistant DHFR-variant, and wherein the host cell is cultured with the antifolate. 10. The method of claim 9, wherein one or more of the following features are met: (a) the antifolate is methotrexate; And (b) the nucleic acid encoding the antifolate-resistant DHFR variant encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-9. 2. The method of claim 1, wherein the protein is selected from the group consisting of cytokines including the tumor necrosis factors TNF-alpha and TNF-beta and their receptor groups; Renin; Human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipid protein; Alpha-1-antitrypsin; Insulin A-chain and B-chain; Gonadotropin, including follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin, and human chorionic gonadotropin (hCG); Calcitonin; Glucagon; Factor VIIIC, Factor IX, Factor VII, Coagulation Factor, Tissue Factor and Bilevelrant Factor; A anti-coagulation factor comprising protein C; Atrial Sodium Excretion Promoter; Pulmonary surfactants; A plasminogen activator comprising a plasminogen activator of europine kinase, human urine and tissue type; Bombesh Shin; Thrombin; Hematopoietic growth factors; Enkephalinae; Human macrophage inflammatory protein; Serum albumin, including human serum albumin; Mullerian-inhibiting substances; Lt; / RTI &gt; A-chain and B-chain; Prolaxin; Mouse hypertonic tropin-related peptides; Vascular endothelial growth factor; Hormone or growth factor receptor; Integrin; Proteins A and D; Rheumatic factor; Bone-derived neurotrophic factor, neurotrophin-3, -4, -5, -6 and nerve growth factor-beta; Platelet derived growth factor; Fibroblast growth factor; Epidermal growth factor; A transforming growth factor comprising TGF-alpha and TGF-beta; Insulin-like growth factor-I and -II; Insulin-like growth factor binding protein; CD proteins including CD-3, CD-4, CD-8 and CD-19; Erythropoietin (EPO); Bone inducer; Immunotoxin; Osteogenic protein; Interferons including interferon-alpha, -beta and-gamma; Colony stimulating factor (CSF's) comprising M-CSF, GM-CSF and G-CSF; Interleukins (IL's) comprising IL-1 to IL-12; Superoxide dimutase; T-cell receptors; Surface membrane protein; Decay accelerating factor; Antibodies and immunoconjugates; Glycophorin A; MUC1. &Lt; / RTI &gt; The method of claim 1, wherein the protein is an antibody or fragment thereof. 13. The method of claim 12, wherein the antibody is selected from the group consisting of an antibody to Ganglioside GD3, an antibody to a human interleukin-5 receptor alpha-chain, an antibody to HER2, an antibody to CC kemokin receptor 4, A human antibody, an antibody against neuroblastoma, an antibody against MUC1, and an antibody against epidermal growth factor receptor. 13. The method of claim 12, wherein the antibody is selected from the group consisting of Pankomab, Muromomab, Daclizumab, Basiliximab, Abciximab, Rituximab Herceptin, Gemtuzumab, Alemtuzumab, Ibritumomab, Cetuximab, Bevacizumab, Tositumomab, and the like. Ephthalimab, Eculizumab, Epratuzumab, Omalizumab, Efalizumab, Adalimumab, Kampatsu-1H, and others. (Campath-1H), C2B8, Panorex, BrevaRex, Simulect, Antova, OKT3, Zenapax, ReoPro, Synagis ), Ostavir, Protovir, OvaRex, Vitaxin, anti-CCK moiety receptor 4 antibody KM2160, and anti-neuroblastoma antibody chCE7 &Lt; / RTI &gt; 13. The method according to claim 12, wherein the antibody is selected from the group consisting of an antibody against MUC1 comprising pancomap, panco 1 and panco 2, an antibody against HER2 comprising HERCceptin, an antibody against EGFR comprising cetuximab How to do it. The method of claim 1, (i) is fused to another peptide or polypeptide sequence, (ii) a protein sequence selected from the group consisting of cytokines, co-stimulatory factors, toxins, antibody fragments from other antibodies, multimerization sequences, sequences for detection, purification, secretion or stabilization, localization signals or linkers An antibody fused to another protein sequence, (iii) an antibody or antibody fragment that is coupled to an antibody or antibody fragment to an effector molecule that mediates the therapeutic effect. (a) introducing one or more nucleic acids derived from the cell line K562 [ATCC CCL-243] and encoding the protein or at least a portion thereof in two or more different host cells providing different glycosylation patterns, wherein the cell line K562 [ATCC CCL -243] is an infinite proliferating human blood cell: (b) culturing the two or more different host cells, wherein each different host cell produces a protein composition having a glycosylation pattern that is different from the glycosylation pattern produced by the different host cells; (c) isolating said expressed protein composition having a different glycosylation pattern from two or more different host cells; And (d) selecting a host cell that produces a protein composition having the following glycosylation characteristics by selecting said host cell to produce a protein composition having glycosylation characteristics as defined in (i) to (v): As a method, The host cell is a cell or cell line derived from NM-H9D8 [DSM ACC2806], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856] and GT- &Lt; / RTI &gt; (i) does not include detectable NeuGc; (ii) does not contain detectable terminal Gal Gal 1-3Gal; (iii) at least 2% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iv) alpha 2-6 bound NeuNAc; And (v) Expression CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 15% higher. A protein molecular composition obtainable by the production method according to claim 1, wherein the protein in the protein molecular composition has the following glycosylation characteristics: (i) does not include detectable NeuGc; (ii) alpha 2-6 bound NeuNAc; And  (iii) Expression of CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 15% higher. 19. The protein composition of claim 18, having one or more of the following characteristics: (i) does not contain detectable terminal Gal alpha 1-3Gal; (ii) at least 2% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iii) at least 5% carbohydrate structure of at least one particular carbohydrate chain or an entire carbohydrate unit at a specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iv) CHOdhfr- [ATCC No. Compared to the same amount of protein molecules in one or more protein molecular compositions of the same protein molecule isolated from the same protein molecule isolated from a protein carbohydrate structure or a specific glycosylation site of a protein molecule of the protein molecule composition, Structure, an increased degree of sialylation with a NeuNAc amount of at least 20% greater; (v) protein molecules of the protein molecular composition lacking fucose include at least 50% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glycosylation site of the white matter molecule; And (DSM ACC2606] or NM-D4 [DSM ACC2605] cells have higher degree of sialylation than those obtained in (vi) NM-F9 [DSM ACC2606] or NM- But only 50 to 60% of the degree of sialylation obtained for the host cells. 19. A protein molecule composition according to claim 18 having one or more of the following features: For the whole carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. Has a degree of galactosylation of increased galactose as compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CRL-9096; For the whole carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096] at least 5% higher in the amount of G2 structure compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from the same protein molecule; For the whole carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. CRL-9096], at least 5% lower than the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from the same protein molecule; For the whole carbohydrate structure or the carbohydrate structure at one particular glycosylation site of the protein molecule among the protein molecules of the protein molecule composition, CHOdhfr- [ATCC No. Comprising at least 5% lower amount of fucose as compared to the same amount of protein molecule in at least one protein molecule composition of the same protein molecule isolated from CRL-9096; At the time of expression, CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 20% higher than that of the native structure; At the time of expression, CHOdhfr- [ATCC No. As compared to the same amount of protein molecules in one or more protein molecule compositions of the same protein molecule isolated from the same glycosylation site of the protein molecule, Carbohydrate chains bound by charged N-glycosides in excess of at least 20% of the total carbohydrate unit; At least one specific carbohydrate chain at the specific glycosylation site of the protein molecule in the protein molecules of the protein molecule composition lacking fucose or at least 10% carbohydrate structure of the entire carbohydrate unit; At least one particular carbohydrate chain at the specific glycosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc or at least 5% carbohydrate structure of the entire carbohydrate unit; A G2 structure in excess of 35% on a carbohydrate structure or a total carbohydrate structure at a particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition; Less than 22% of the G0 structure on the carbohydrate structure or on the total carbohydrate structure at one particular glycosylation site of the protein molecule of the protein molecule composition; The cell line CHOdhfr- [ATCC No. CRL-9096] as compared to one or more protein molecule compositions of the same protein molecule; The cell line CHOdhfr- [ATCC No. CRL-9096] compared to one or more protein molecular compositions of the same protein molecule; The cell line CHOdhfr- [ATCC No. CRL-9096] at least 10% higher than the yield of one or more protein molecular compositions of the same protein molecule at the time of expression; The cell line CHOdhfr- [ATCC No. CRL-9096] at least 10% higher than the activity of one or more protein molecule compositions of the same protein molecule; Wherein the antibody molecule composition comprises the cell line CHOdhfr- [ATCC No. &Lt; / RTI &gt; Has a degree of sialylation that is lower than the degree of sialylation of one or more antibody molecule compositions from the same antibody molecule upon expression in CRL-9096; When the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. Nos. Has a cytotoxicity of increased Fc-mediated cell that is at least 2-fold higher than the cytotoxicity of Fc-mediated cells of one or more antibody molecule compositions from the same antibody molecule upon expression in CRL-9096; And When the protein molecule is an antibody molecule, the cell line CHOdhfr- [ATCC No. Nos. CRL-9096] has at least 50% higher antigen-mediated binding or Fc-mediated binding than binding of one or more antibody molecule compositions from the same antibody molecule. 19. The method of claim 18, wherein the protein is selected from the group consisting of cytokines and their receptor groups including tumor necrosis factors TNF-alpha and TNF-beta; Renin; Human growth hormone and bovine growth hormone; Growth hormone releasing factor; Parathyroid hormone; Thyroid stimulating hormone; Lipid protein; Alpha-1-antitrypsin; Insulin A-chain and B-chain; Gonadotropin hormones including follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin, and human chorionic gonadotropin (hCG); Calcitonin; Glucagon; Factor VIIIC, Factor IX, Factor VII, Coagulation Factor, Tissue Factor and Bilevelrant Factor; A anti-coagulation factor comprising protein C; Atrial Sodium Excretion Promoter; Pulmonary surfactants; A plasminogen activator comprising a plasminogen activator of europine kinase, human urine and tissue type; Bombesh Shin; Thrombin; Hematopoietic growth factors; Enkephalinae; Human macrophage inflammatory protein; Serum albumin, including human serum albumin; Mullerian-inhibiting substances; Lt; / RTI &gt; A-chain and B-chain; Prolaxin; Mouse hypertonic tropin-related peptides; Vascular endothelial growth factor; Hormone or growth factor receptor; Integrin; Proteins A and D; Rheumatic factor; Bone-derived neurotrophic factor, neurotrophin-3, -4, -5, -6 and nerve growth factor-beta; Platelet derived growth factor; Fibroblast growth factor; Epidermal growth factor; A transforming growth factor comprising TGF-alpha and TGF-beta; Insulin-like growth factor-I and -II; Insulin-like growth factor binding protein; CD proteins including CD-3, CD-4, CD-8 and CD-19; Erythropoietin (EPO); Bone inducer; Immunotoxin; Osteogenic protein; Interferons including interferon-alpha, -beta and-gamma; Colony stimulating factor (CSF's) comprising M-CSF, GM-CSF and G-CSF; Interleukins (IL's) comprising IL-1 to IL-12; Superoxide dimutase; T-cell receptors; Surface membrane protein; Decay accelerating factor; Antibodies and immunoconjugates; Glycophorin A; MUC1. &Lt; / RTI &gt; 19. The composition of claim 18, wherein said protein is an antibody or fragment thereof. 23. The composition of claim 22, wherein the antibody is an IgG class. 23. The composition of claim 22, wherein the antibody is human, humanized or chimeric IgG bearing a human Fc region. 23. The protein molecule composition of claim 22 comprising at least one sugar form of an antibody molecule having at least one carbohydrate chain attached to another glycosylation site of the antibody molecule than the amino acid Asn-297 in the second domain of the Fc region. 23. The method of claim 22, wherein the antibody has at least one N-glycosylation site or at least one O-glycosylation site having an amino acid sequence of Asn-Xaa-Ser / Thr at the sequence of the Fab region, Amino acid residues and amino acids. 23. The method of claim 22, (a) an antibody molecule comprising at least one carbohydrate chain attached to another glycosylation site of the antibody molecule at a second domain of the Fc portion than the amino acid Asn-297, wherein the antibody molecule composition expresses the cell line CHO, CHOdhfr-, BHK , Antibody molecules of the same antibody molecule isolated from NS0, SP2 / 0, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], PerC.6 or mouse hybridomas, An antibody having an extended serum half-life or bioavailability; or (b) one or more carbohydrate chains attached to one or more N-glycosylation sites or one or more O-glycosylation sites in the sequence of the Fab region of the antibody molecule, wherein the antibody molecule comprises a cell line CHO, An antibody molecule having an extended serum half-life or bioavailability as measured in one or more mammals than an antibody molecule composition of the same antibody molecule isolated from CHOdhfr-, BHK, NSO, SP2 / 0, PerC.6 or a mouse hybridoma &Lt; / RTI &gt; 23. The method of claim 22, (i) is fused to another peptide or polypeptide sequence, (ii) a protein sequence selected from the group consisting of cytokines, co-stimulatory factors, toxins, antibody fragments from other antibodies, multimerization sequences, sequences for detection, purification, secretion or stabilization, localization signals or linkers An antibody fused to another protein sequence, (iii) a protein molecule conjugated to an effector molecule that mediates a therapeutic effect. 23. The method of claim 22, wherein the antibody is selected from the group consisting of an antibody to Ganglioside GD3, an antibody to a human interleukin-5 receptor alpha-chain, an antibody to HER2, an antibody to CC chemokine receptor 4, An antibody against MUC1, an antibody against epidermal growth factor receptor, and a protein molecule composition selected from the group consisting of an antibody against human antibody, an antibody against neuroblastoma, an antibody against MUC1, and an antibody against epidermal growth factor receptor. 23. The method of claim 22, wherein the antibody is selected from the group consisting of Pancomaps, Mylomops, Daclizumaps, Bacillus sephaps, Abcissimaps, Rituximaps, Herceptin, Gemtuzumaps, Alemtuzumaps, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, Cobb, From the group consisting of Simullect, Antova, OKT3, Jenapax, Leopro, Sinagis, Ostivir, Protovir, Obarex, Vitaxin, Anti-CCmokin receptor 4 antibody KM2160, and Anti-Neuroblastoma antibody chCE7 Selected protein molecular composition. 23. The composition of claim 22, wherein the composition comprises at least one antibody molecule that binds to a MUC1 epitope comprising an amino acid DTR of an extracellular tandem repeating region. 32. The composition of claim 31, wherein said antibody is selected from antibody pancomaps, antibody panco 1 and antibody panco 2. 19. The method of claim 18, (i) an antibody to a vascular endothelial growth factor; (ii) an antibody against HER2; (iii) an antibody against CD20; (iv) gonadotropin; And (iv) a coagulation factor. As infinitely proliferating human blood cells derived from cell line K562 [ATCC CCL-243] capable of producing a protein molecular composition having the following glycosylation characteristics, The endogenous proliferating human blood cells are selected from the group consisting of NM-H9D8 [DSM ACC2806], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856] and GT- 2X [DSM ACC2858] &Lt; / RTI &gt; infinitely proliferating human blood cells selected from the group consisting of: (i) does not include detectable NeuGc; (ii) alpha 2-6 bound NeuNAc; And  (iii) Expression of CHOdhfr- [ATCC No. CRL-9096], the total carbohydrate structure or the number of carbohydrates at one particular glycosylation site of the protein molecule in the protein molecule of the protein molecule composition, as compared to the same amount of protein molecule in one or more protein molecular compositions of the same protein molecule Structure, with an amount of NeuNAc of at least 15% higher. 35. The infant proliferating human blood cell of claim 34 wherein the protein molecular composition further has one or more of the following characteristics: (i) does not contain detectable terminal Gal alpha 1-3Gal; (ii) at least 2% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iii) at least 5% carbohydrate structure of at least one particular carbohydrate chain or an entire carbohydrate unit at a specific glucosylation site of the protein molecule in the protein molecules of the protein molecule composition containing the bisecting GlcNAc; (iv) CHOdhfr- [ATCC No. Compared to the same amount of protein molecules in one or more protein molecular compositions of the same protein molecule isolated from the same protein molecule isolated from a protein carbohydrate structure or a specific glycosylation site of a protein molecule of the protein molecule composition, Structure, an increased degree of sialylation with a NeuNAc amount of at least 20% greater; (v) protein molecules of the protein molecular composition lacking fucose include at least 50% of the carbohydrate structure of at least one particular carbohydrate chain or the entire carbohydrate unit at the specific glycosylation site of the white matter molecule; And (DSM ACC2606] or NM-D4 [DSM ACC2605] cells have higher degree of sialylation than those obtained in (vi) NM-F9 [DSM ACC2606] or NM-D4 [DSM ACC2605] And only 50 to 60% of the degree of sialylation obtained for the host cell derived from cell line K562 [ATCC CCL-243]. 36. The method of claim 34, wherein the cell or cell strain is selected from the group consisting of NM-H9D8 [DSM ACC 2806], NM-H9D8-E6 [DSM ACC 2807], NM-H9D8-E6Q12 [DSM ACC 2856] Infinitely proliferating human blood cells. 35. The method of claim 34, further comprising one or more nucleic acids encoding at least a portion of a protein molecule, and at least one nucleic acid sequence encoding at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-9. Infinitely proliferating human blood cells containing nucleic acids. A group consisting of cells or cell lines derived from them, and a group consisting of NM-H9D8-E6 [DSM ACC 2858], NM-H9D8 [DSM ACC 2806], NM-H9D8-E6 [DSM ACC 2807] Lt; / RTI &gt; human blood cells. 35. An infinite proliferating human blood cell according to claim 34, comprising at least one nucleic acid encoding them to produce a protein molecule or at least a portion thereof. A method for producing a protein molecular composition comprising: (a) introducing one or more nucleic acids encoding at least a portion of said protein into a host cell derived from cell line K562 [ATCC CCL-243]; (b) culturing the host cell under conditions that allow the production of the protein molecular composition; And (c) separating the protein molecular composition, The host cell H9D8-E6Q12 [DSM ACC 2856], and cells or cell lines derived therefrom. A protein molecular composition obtained by the production method according to claim 40. delete delete delete delete delete delete

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